RIRDC Completed Projects in 1997-1998 and Research in ...€¦ · A/Prof David Farrell (07) 3365...

RIRDC Completed Projects in 1997-1998 and Research in Progress as at June 1998

3.1 Chicken Meat & Joint Chicken Meat & Egg Industry Projects

August 1998 RIRDC Publication No 98/91

Sub-program 3.1 - Chicken Meat and Joint Chicken Meat and Egg Industry Projects


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© 1998 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 57818 4 ISSN 1440-6845 "RIRDC Completed Projects in 1997-98 and Research in Progress as at June 1998 - 3.1 Chicken Meat & Joint Chicken Meat & Egg Industry Projects" Publication No 98/91 The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Communications Manager on phone 02 6272 3186.

RIRDC Chicken Meat Research Manager Dr Vivien Kite RIRDC Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: (02) 9929 4077 Fax: (02) 9925 0627 Email: [email protected] RIRDC Publications Manager Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: (02) 6272 3186 Fax: (02) 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au

Published in December 1998 Printed on environmentally friendly paper by Union Offset



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FOREWORD This year RIRDC has produced Research in Progress, June '98, which contains short summaries of continuing projects as well as those that were completed during 1997-98 for all of the Corporation’s 21 program areas. The complete report on all the programs is only available in electronic format on our website at http://www.rirdc.gov.au The following report is a hardcopy extract covering Sub-Program 3.1. It contains all entries from continuing and completed Chicken Meat research projects funded by RIRDC, as well as projects jointly funded through the Chicken Meat and Egg Industry programs. This program aims to support increased profitability and sustainability in the chicken meat industry through improvements in efficiency, product quality and market size and through the adoption of enlightened management practices. This report is the newest addition to our extensive catalogue of over 200 research reports, videos and CD-Roms of projects supported by RIRDC. Please contact us for the latest publications catalogue or view it on our website. Peter Core Managing Director Rural Industries Research and Development Corporation



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CONTENTS

COMPLETED PROJECTS

PROJECT No

PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE No

CSK-2CM Growth pattern and reproduction in meat chicken female lines

Dr Jeff Downing (02) 4655 0600 CSIRO Division of Animal Production

1

DAS-10CM Factors influencing the nutritive value of lupins

Mr Robert Hughes (08) 8303 7788 South Australian Research & Development Institute

1

IMVS-6CM Epidemiological studies of Salmonella using serotyping, plasmid analysis, chromosomal and PCR analysis

Dr Michael Heuzenroeder

(08) 8222 3275 Institute of Medical & Veterinary Science

2

RMIT-5CM Differentiation of virulent and avirulent Campylobacter jejuni and Campylobacter colistrains isolated from humans and chickens.

Dr Victoria Korolik (03) 9660 2976 Royal Melbourne Institute of Technology

3

UNE-54A Increasing efficiency of lean tissue deposition in broiler chickens

Dr Mingan Choct (02) 6773 5121 University of New England 4

UQ-64A The metabolisable energy of ‘viscous’ and problem grains measured at two sites and methods of prediction of grain samples likely to respond to enzyme additions

A/Prof David Farrell (07) 3365 2051 University of Queensland 4

US-67CM Determination of true amino acid digestibility of feedstuffs

A/Prof Wayne Bryden (02) 4655 0658 University of Sydney 5

RESEARCH IN PROGRESS

PROJECT No


ANU-21A Junior Research Fellowship – Ms Lisa Alleva Dr Karen Ovington (02) 6249 4941 Australian National University 7 CSJ-2A Control of ovarian development and function

in meat chicken females Dr Jeff Downing (02) 4655 0600 CSIRO Division of Animal

Production 7

DAN-40CM On farm comparison of computer controlled tunnel ventilated and naturally ventilated broiler sheds

Mr Gerry Bolla (02) 4328 0317 NSW Agriculture 7

DAN-142A Production of distance learning materials for the poultry industry – stage III continued

Mr Geoff Creek (02) 6953 0299 NSW Agriculture 8

DAN-158A Infectious proventriculitis and stunting syndrome of broiler chickens

Dr Rod Reece (02) 4640 6309 NSW Agriculture 8

DAV-120A The effect of Salmonella Sofia on intestinal infection with other salmonellae in chickens

Dr Colin Wilks (03) 9333 1200 Dept of Natural Resources & Environment (Vic)

9

DAW-74A The control of big liver and spleen disease (BLS)

Dr Trevor Ellis (08) 9368 3631 Agriculture Western Australia 9

IMVS-1A Molecular basis of benign colonisation of Salmonella Sofia in chickens

Dr Michael Heuzenroeder

(08) 8222 3275 Institute of Medical & Veterinary Science

10

RMI-7A Use of avirulent Campylobacter jejuni strains to control poultry-derived Campylobacter food poisoning

Dr Victoria Korolik (03) 9660 2110 Royal Melbourne Institute of Technology

10

SGH-5A Managing structural change in the Australian poultry industry

Mr Terry Larkin (02) 9955 2722 S. G. Heilbron Consulting Pty Ltd

11

UMO-12CM Identification of in vivo expressed antigens of Pasteurella multocida

Dr Ben Adler (03) 9905 4815 Monash University 11

UNE-53A The “new season grains” phenomenon and the role of the endogenous glycanases in grains for their nutritive value in poultry

Dr Mingan Choct (02) 6773 5121 University of New England 11

UNE-64A Postgraduate scholarship - Andreas Kocher: Increasing the nutritive value of grain legumes for poultry by use of more efficacious enzyme systems

Dr Mingan Choct

(02) 6773 5135 University of New England 12

UNS-10A Defined probiotic preparations for competitive exclusion of enteropathogens from poultry

Dr Julian Cox (02) 9385 5665 University of New South Wales 12

UNS-11A Odour and ammonia emission reduction from chicken broiler growout farms

Mr John Jiang (02) 9385 5452 University of New South Wales 13

UQ-57A Development of a new fowl pox vaccine strain

Prof Wayne Robinson (07) 3365 2565 University of Queensland 13

US-34A Development of DNA vaccines and techniques for cytokine manipulation for improved mucosal immunity in chickens

Prof Alan Husband (02) 9351 3127 University of Sydney 14



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RESEARCH IN PROGRESS

PROJECT

No PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE

No US-35A The development of a model of abnormal

bone development in broiler chicken as an aid in the assessment of welfare


US-38A Broiler feed formulation based on digestible amino acids


US-44A Manipulation of lean tissue deposition by altering the sensitivity of tissues to insulin


UTS-1A Expression of Eimeria genes in Toxoplasma Dr John Ellis

(02) 9330 4161 University of Technology, Sydney

15

UTS-2A A maternally delivered vaccine against coccidiosis in broiler chickens

Dr Nicholas Smith (02) 9514 4013 University of Technology, Sydney

16

COMPLETED JOINT PROJECTS

PROJECT No


CSK-3A Infectious bursal disease virus (IBDV): To determine if current vaccination strategies prevent the emergence of very virulent IBDV strains in Australia

Dr Jagoda Ignjatovic

(03) 9227 5000 CSIRO Division of Animal Health

17

DAQ-30CM and DAQ-28E

Testing and improving the nutritional value of grain legumes for layers and broilers

Mr Paul Mannion (07) 3824 3081 Dept of Primary Industries (Qld)

17

DAQ-200A Rapid and specific detection of avian bacterial pathogens

Dr Pat Blackall (07) 3362 9498 Dept of Primary Industries (Qld)

18

DAQ-226A Sabbatical studies – Dr Mazhur Khan: Development of molecular tests for serovar-specific identification and typing of Haemophilus paragallinarum

Dr Pat Blackall (07) 3362 9498 Dept of Primary Industries (Qld)

19

DAV-115A Development of standard MDV challenge viruses in cell culture

Dr Robin Condron (03) 9217 4200 Dept of Natural Resources & Environment (Vic)

20

UQ-52A A comparison of total and digestible amino acids in diets for broilers and layers

Dr David Farrell (07) 3824 3081 University of Queensland 20

US-62CM and US-64CM/ US-44E

Novel strategies for improved mucosal immunity in chickens - application to control of Salmonellosis; and Junior Research Fellowship – Ms Wendy Muir

Prof Alan Husband (02) 9351 7130 University of Sydney 21

JOINT RESEARCH IN PROGRESS

PROJECT No


CSA-2A Diagnostic tools for detection of vvIBDV strains and characterisation of Australian variants

Dr Jagoda Ignjatovic

(03) 9227 5000 CSIRO Division of Animal Health

23

DAN-130A Field evaluation of mass vaccination techniques using V4 and heat resistant V4 (HRV4) Newcastle disease virus vaccine strains on caged layers

Mr George Arzey (02) 4640 6333 NSW Agriculture 23


Improved control strategies for Marek’s disease

Dr Peter Young (07) 3362 9400 Dept of Primary Industries (Qld)

24


Marek’s disease challenge trials Dr Peter Young (07) 3362 9400 Dept of Primary Industries (Qld)

24

DAQ-212A Marek’s disease challenge trial 4 Dr Peter Young (07) 3362 9400 Dept of Primary Industries (Qld)

24

DAQ-215A Attenuation and characterisation of Eimeria species for use in a living vaccine for avian coccidiosis

Dr Wayne Jorgensen (07) 3362 9455 Dept of Primary Industries (Qld)

25

DAQ-228A Raw soybeans selected for low trypsin inhibitor activity for poultry diets

Dr Rider Perez-Maldonado

(07) 3824 3081 Dept of Primary Industries (Qld)

25

DAV-125A Marek’s disease vaccine seed attenuation and challenge trial

Dr Robin Condron (03) 9217 4200 Dept of Natural Resources & Environment (Vic)

26

DAV-145A Three vaccine trials on Marek’s disease Dr Robin Condron (03) 9217 4200 Dept of Natural Resources & Environment (Vic)

26



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JOINT RESEARCH IN PROGRESS

PROJECT

No PROJECT TITLE RESEARCHER PHONE ORGANISATION PAGE

No GRD-1A Improving feed grains quality Dr John Black (02) 4753 6231 Grains Research &

Development Corporation 26

RMIT-4CM and RMIT-12E

Development of Marek’s disease Type 1 vaccine

Prof Greg Tannock (03) 9925 3088 Royal Melbourne Institute of Technology

27

RMI-6A The development of effective immunisation strategies against Marek’s disease


27

RMI-8A Characterisation of very virulent Australian isolates of Marek's disease virus


28

UM-33A Development of improved serological diagnosis of Mycoplasma synoviae

Dr Glenn Browning (03) 9344 7342 University of Melbourne 28

UMU-21A Intestinal spirochaete infections in chickens in eastern Australia

A/Prof David Hampson (08) 9360 2287 Murdoch University 29

UNS-9A Evaluation of the VICAM Screen/Verify system for detection of Salmonella Enteritidis in poultry flocks

Dr Julian Cox (02) 9385 5665 University of New South Wales 29

US-51A Total and digestible tryptophan content of feedstuffs for poultry


Page 1 Sub-program 3.1 - Chicken Meat and Joint Chicken Meat and Egg Industry Projects


3.1: CHICKEN MEAT COMPLETED PROJECTS

Project Title Growth pattern and reproduction in meat chicken female lines RIRDC Project No: CSK-2CM Researcher: Dr Jeff Downing and Dr Rhys

D Roberts Organisation: CSIRO Division of Animal

Production c/- Dept of Animal Science University of Sydney Werombi Road CAMDEN NSW 2570

Contacts: Phone: (02) 4655 0600 Fax: (02) 4655 0693 E-mail [email protected] Objectives • To improve the understanding of ovarian growth

mechanisms in the hen by examining the effects of growth factors using a tissue culture system designed to represent the growing ovary.

• To identify pathways that may be exploited to cause an improvement in reproductive efficiency of broiler hens.

Background Long-term selection for meat production has resulted in lower reproductive performance of broiler breeder females associated with disrupted ovarian function (larger number of ovarian follicles grow but fewer eggs are laid). This project sought to understand the physiology of this problem so that improvements to ovarian function can be made by physiological means, while leaving the large genetic gains in broiler breeder lines intact. Research An ovarian cell culture system was established, capable of measuring ovarian cell growth (as DNA synthesis). The effects of the growth factors IGF-1 and IGF-2 (insulin-like growth factors 1 and 2), EGF (epidermal growth factor), TGFα (transforming growth factor α), FGF-1 and FGF-2 (fibroblast growth factors 1 and 2) and GH (growth hormone) on DNA synthesis of cultured ovarian (granulosa and thecal) cells was investigated. The growth effects of factors added individually and in combinations (IGF-1, TGFα and FGF2) were compared. An investigation of the IGF system was undertaken, particularly focussing on the presence, effects and purification of chicken ovarian IGF binding proteins (IGFBPs). Outcomes All tested growth factors stimulated growth of chicken ovarian cells, however the potency of each factor was different. GH had no direct effect on the growth of

ovarian cells. Growth factor combinations were many times more potent than factors applied singly. Ovarian cells produce IGFBPs that can modify the actions of IGF-1 on these cells. IGF-1, in turn, can modify the production of IGFBPs by granulosa cells. Purification of ovarian IGFBPs was problematic. Implications Growth factors, including the IGFs, are potent stimulators of ovarian cell growth. The IGFs may mediate the actions of GH. Growth factor synergy indicates a complex mechanism for control of the growth of ovarian follicles. The existence of functional ovarian IGFBPs indicates the existence of an autocrine/paracrine IGF system which may be subject to artificial control by IGFBP-like compounds in vivo. Publications Roberts, R.D. and Gordon, B.M. (1994) The effects of

growth factors on chicken ovarian cells in vitro. Proc. 1994 Aust. Poult. Sci. Symp. 7: 65-71.

Roberts, R.D. and Ellis, R.C.L. (1995) Growth factor synergy in chicken ovarian cell growth. Proc. 27th Ann. Conf. Aust. Soc. Reprod. Biol. pp 138.

Project Title Factors influencing the nutritive value of lupins RIRDC Project No: DAS-10CM Researcher: Mr Robert Hughes, Dr Robert

van Barneveld, Mr Andreas Kocher and Dr Mingan Choct

Organisation: South Australian Research & Development Institute Pig and Poultry Production Institute University of Adelaide, Roseworthy Campus

ROSEWORTHY SA 5371 Contacts: Phone: (08) 8303 7788 Fax: (08) 8303 7977 E-mail: [email protected] Objectives • To collect a range of samples of Lupinus

angustifolius and Lupinus albus cultivars with associated information on growing conditions.

• To determine AME and analyse the composition of each sample.

• To examine the effects of dietary addition of hulls, soluble and insoluble non-starch polysaccharides (NSP) and oligosaccharides, and develop strategies to minimise any anti-nutritive effects.

Background Although lupins are widely used as a monogastric protein source, uncertainty surrounds the effects of high dietary levels of oligosaccharides and NSP, the value of additional processing such as de-hulling and the cost-effectiveness of enzyme products. Strategies to overcome these problems are needed in order that greater use can be

mailto:[email protected]




made of lupins in broiler diets with anticipated reductions in feed costs. Research The nutritive values of Australian sweet lupin cultivar Gungurru and white lupin cultivar Kiev mutant were assessed in a series of apparent metabolisable energy studies on commercial broiler chickens housed in metabolism cages. Outcomes Modern cultivars of Australian lupins are valuable alternative sources of protein and energy for inclusion in broiler diets. Large differences in nutritive value due to species, season and locality were evident. The seed coats of these species do not contain anti-nutritive factors but did have an energy dilution effect. Feed enzyme products failed to yield consistent results in short-term AME studies on different breeds of broiler chickens in metabolism cages. Implications Wholeseed of Gungurru lupin can be included at 200 g/kg in a wheat-based diet and at 300 g/kg in a maize-based diet without deleterious effects on growth or feed efficiency. Excreta moisture is increased by inclusion of wholeseed of Gungurru lupin at 200 g/kg in wheat-based and maize-based diets. Enzyme technology for degrading lupin NSP and oligosaccharides needs development. Publications Annison, G., Hughes, R.J. and Choct, M. (1996) Effects

of enzyme supplementation on the nutritive value of de-hulled lupins. Brit. Poult. Sci. 37: 157-172.

Hughes, R.J., Kocher, A. and Choct, M. (1998) Nutritive value of lupins for broilers. Proc. 1998 Aust. Poult. Sci. Symp. 10: 140-143.

Project Title Epidemiological studies of Salmonella using serotyping, plasmid analysis, chromosomal and PCR analysis RIRDC Project No: IMVS-6CM Researcher: Dr Michael W Heuzenroeder Organisation: Institute of Medical &

Veterinary Science PO Box 14 Rundle Mall ADELAIDE SA 5000

Contacts: Phone: (08) 8222 3275 Fax: (08) 8222 3543

E-mail: [email protected] Objectives • To continue to monitor and type, by conventional

serotyping and other typing methods, Salmonella isolates submitted by the poultry industry.

• To develop universal DNA based typing methods for Salmonella sub-typing.

• To survey genes that are implicated in the virulence of Salmonella in serovars of interest to the industry.

Background Salmonellosis associated with poultry is a problem worldwide for both producers and consumers. Salmonella are traditionally classified into serovars on the basis of the O and H antigens. It is necessary for the purposes of organism tracing and epidemiology to further subdivide Salmonella isolates. Classical methods, such as phage typing, though useful do not always give sufficient discrimination. This has lead to the development of molecular typing methods that are based upon DNA technology. Salmonella Sofia represented over 78% of Salmonella isolated from chickens in 1995. Despite its widespread colonisation of chickens, it is rarely represented in the serovars isolated from humans. The conclusion is that Salmonella Sofia is not a significant pathogen for humans or chickens. This avirulence may be due to the absence of virulence genes. The DNA sequence data of these genes is available and can be used in PCR or other molecular methods to detect these genes and possibly explain the avirulence of S. Sofia. Research Serovars in poultry have not changed significantly in the past three years. S. Sofia continues to be the major isolate in commercial broiler flocks in Australia, but almost absent in the human population. There appears to be no reliable ‘universal’ method of molecular typing available at present. PFGE is rapidly becoming the method of choice in many laboratories, however it is of limited use in highly clonal serovars. Phage typing remains the technique of first choice for organism typing, however when interpreting results it must be remembered that bacterial populations are dynamic entities. The invH gene, and to a lesser extent other genes within the inv cluster, are often incomplete and may have some role in the avirulent phenotype of S. Sofia. The absence of spv plasmid borne genes in Sofia is universal. It is possible that a large plasmid in S. Sofia may exclude the acquisition of virulence factors from virulent serovars found in chickens. Outcomes • The continued monitoring of industry flocks over the

past three years and organism tracing when outbreaks occur.

• Development of a number of molecular typing methods and a partial understanding of the significance of phage type conversion when tracing organisms.

• Partial explanation of the avirulence of S. Sofia. Implications Continued strong epidemiological and scientific evidence indicates that Salmonella Sofia, the most common Salmonella colonising meat chickens, is avirulent. The mechanism of this avirulence is unknown, but it may be a combination of a deficiency in one or more of the genes




required for virulence. This evidence could be used in public awareness campaigns that S. Sofia is harmless and may be beneficial in excluding more virulent serovars. Continual monitoring of chicken flocks for changes in the distribution and serovar type provides clear evidence to the consumer that the industry takes the issue of product safety extremely seriously. Project Title Differentiation of virulent and avirulent Campylobacter jejuni and Campylobacter coli strains isolated from humans and chickens RIRDC Project No: RMIT-5CM Researcher: Dr Victoria Korolik and Prof

Peter J Coloe Organisation: Royal Melbourne Institute of

Technology Dept of Applied Biology & Biotechnology 124 LaTrobe Street MELBOURNE VIC 3000


E-mail: [email protected] Objectives • To develop a rapid, simple detection method and an

identification scheme for Campylobacter spp. of medical and veterinary importance.

• To develop methods to differentiate virulent from avirulent Campylobacter spp.

• To identify and replace strains potentially pathogenic to humans in chicken flocks with a colonising strain, adapted to chickens but avirulent for people.

Background Campylobacter species such as C. jejuni and C. coli are recognised as major causes of acute gastroenteritis worldwide in humans. Consumption of contaminated foods, including poultry, has been recognised as a source of the disease in humans. However it is possible that not all Campylobacter strains that may inhabit chickens may be capable of producing disease in humans. Hence, it is important that tests be developed that can distinguish potentially pathogenic strains from non-pathogenic strains and that strategies be developed to reduce the frequency of virulent strains in chickens. Research A DNA probe was constructed which can distinguish C. jejuni strains into two subgroups based on a DNA polymorphism; a two-band hybridisation profile typical of the majority of strains of chicken origin, or a single-band profile typical of the majority of strains of human origin. Using this DNA probe, it was shown for the first time that there are definite genetic differences between those strains of Campylobacter spp. that colonise chickens and those that are isolated from human disease. Research was also undertaken to establish whether there

are differences between strains of C. jejuni in their ability to colonise chickens. Outcomes Outcomes of this work were twofold: • A molecular DNA probe capable of distinguishing

between the majority of the Campylobacter jejuni stains isolated from chickens and those isolated from human patients with campylobacteriosis was developed. The probe has been tested using a large number of C. jejuni strains from various sources in Australia, USA and UK. Approximately 70% of Australian chicken isolates of C. jejuni can be distinguished from human isolates. For USA isolates from chickens it is greater then 90%, and for UK isolates, 60%. The difference in the figures may be due to the differences in chicken farming methods between the countries.

• C. jejuni and C. coli strains isolated from chickens, and those isolated from human patients with campylobacteriosis, were tested for their ability to colonise two week old chicks. Three distinct types of strains were identified: 1. strains unable to establish persistent colonisation in the chicken intestinal tract; 2. strains capable of establishing persistent colonisation, but which are easily displaced by other strains; and 3. one strain which was not only able to colonise chickens, but was able to displace all resident strains. It was shown that an individual strain of C. jejuni can completely dominate the intestinal flora of chickens and displace other Campylobacter strains in a small experimental flock of chickens.

Implications It is essential that a rapid and specific detection and differentiation system be designed to enable separation of disease-causing strains of Campylobacter spp. from avirulent strains. These avirulent strains can form part of the normal flora in chickens. The results of this project will lead to the establishment of a typing scheme for Campylobacter spp. that will be available to the chicken industry to characterise any flock isolates. There is great potential to further develop a molecular typing scheme that will distinguish ‘human virulent’ strains of Campylobacter spp. from ‘normal flora’. Future research will allow a reference laboratory for thermophilic Campylobacter spp. to be established, primarily for chicken isolates but also for human and other animal isolates, where the organism will be identified by comparing with a genetic profile database. The discovery of Campylobacter isolates with varying potential to colonise chickens will enable selection of Campylobacter strains that are super colonisers of the chicken intestinal environment, but which have been characterised as non-pathogenic, for evaluation as potential ‘competitive exclusion’ agents. With further development, such a strain could be used to displace undesirable strains from chickens in commercial flocks and so reduce the possibility of transfer of pathogenic strains to the food chain.




Project Title Increasing efficiency of lean tissue deposition in broiler chickens RIRDC Project No: UNE-54A Researchers: Dr Mingan Choct Organisation: University of New England

School of Rural Science and Natural Resources – Animal Science ARMIDALE NSW 23510


E-mail: [email protected] Objective • To increase the efficiency of lean meat growth in

broiler chickens through nutritional manipulation of protein degradation via the insulin axis.

Background Genetic selection of broiler chickens over the past 40 years has resulted in extremely rapid growth rate. It has also increased carcass fat content. A combination of organic chromium and a branched-chain amino acid, leucine, and some long chain fatty acids may enhance protein deposition, thereby reducing fatness in broiler chickens. Research Two experiments were conducted to examine the effect of graded levels of organic chromium and leucine and four different fat sources on the body composition of broiler chickens. Outcomes Chromium picolinate at 0.5ppm significantly (P<0.05) lowered the carcass fat level. Leucine did not interact with chromium to effect lean growth. Chromium had no effect on bird performance. Dietary leucine above the recommended maintenance level (1.2% of the diet) markedly (P<0.001) reduced the breast muscle yield. The addition of fish oil to broiler diets significantly reduced (P<0.05) the abdominal fat pad weights compared to birds on linseed diets. The amount of fat in the diet (2% or 4%) did not affect body composition. Implications Provisions could be made for chromium levels and some polyunsaturated fatty acids in practical feed formulations to take advantage of their effects on energy utilisation and carcass fat content. Publications Naylor, A. (1997) Honours Thesis, University of New

England: Nutritional Manipulation of Lean Tissue Deposition in Broiler Chickens.

Project Title The metabolisable energy of 'viscous' and problem grains measured at two sites and methods of prediction of grain samples likely to respond to enzyme additions RIRDC Project No: UQ-64A Researcher: A/Prof David J Farrell and Dr

Suzanne Petersen Organisation: University of Queensland

Dept of Agriculture ST LUCIA QLD 4072 Contacts: Phone: (07) 3365 2051 Fax: (07) 3365 1177 E-mail: [email protected] Objectives • To identify and understand why 'viscous' grains

(cereals) and some grain legume seeds sometimes give depressed performance and do not always appear to respond to feed enzymes. Those that do, do not always give improved performance despite an increase in metabolisable energy.

• To develop a method for prediction of problem grain samples and their likelihood of responding to enzyme addition.

Background Some feed grains contain one or more anti-nutritional factors (ANF). In cereal grains these are mainly the non-starch polysaccharides (NSP) such as the arabinoxylans (pentosans) and β-glucans found in the endosperm walls rather than in the outer or bran layers. Not all cereal grains contain NSP but wheat in particular may contain high levels of NSP, particularly the arabinoxylans. As a consequence, when these grains are consumed by poultry some of the NSP goes into solution causing highly viscous gut digesta, particularly in the small intestine. The result of this is reduced nutrient digestion and wet and sticky droppings. The practical consequences can be poor feed conversion ratio, reduced growth rate of broilers, wet litter, hock burn, breast blisters and down-graded carcasses. Feed enzymes have been used to reduce the negative aspects of NSP by degrading them and thereby reducing the viscosity of gut digesta. Since not all wheats and other grains contain anti-nutritional factors in significant amounts, a response to feed enzymes would not always be anticipated. Yet these enzymes are routinely added to wheat-based and other diets at considerable cost, despite the fact that they only really need to be added when the diet contains problem grains. It would be of considerable commercial use to devise a rapid screening method which could identify problem grains prior to their incorporation in diets; a feed enzyme would then be added to alleviate the problem. Research There were three experiments undertaken. All experiments were with broiler chickens grown to 21 days





of age. This the most sensitive period for detecting a response to offending grains and for monitoring a response to feed enzyme additions. In the three experiments, a detailed chemical description was undertaken of the behaviour of wheat, oats and two grain legumes within the bird's digestive system. It was hoped that this information would be used as a basis of an in vitro method which would simulate digestion by the measurement of either the appearance of key metabolites or the disappearance of feed components. Outcomes Four diets were examined in experiment one, these were wheat, oats, sweet lupins and faba beans at high levels of inclusion (250-700 g/kg). AME was considerably lower on all diets at the terminal ileum than in excreta at 22 and 40 days of age suggesting considerable fermentation in the hind gut. This was confirmed by very high levels of the steam volatile fatty acids measured in caecal contents. The wheat sample used was of unusually high viscosity but this did not translate into a low AME value. In the second experiment an examination was undertaken of several published in vitro methods that would predict foregut viscosity and also AME using 24 diets. Inclusion of the wheat samples was 700 g/kg. Relationships between in vivo viscosity and the most promising in vitro method (Bedford's) did not show sufficiently close agreement for the purposes of prediction. Nor did relationships with in vitro viscosity and AME (R2=0.43), digestible energy at the terminal ileum, or gross energy of digesta dry matter at the terminal ileum give useful predictions. AME and gross energy of hindgut digesta, pentosans in feed and hindgut viscosity, and food intake and hindgut viscosity were all examined but again relationships proved to be disappointing. In experiment three, four wheats of different protein levels and extract viscosities were examined. Twelve diets were formulated by adding 0.0, 0.25 or 0.5 g of the exogenous enzyme, Avizyme (Finnfeeds International). Diets were fed to eight birds/cage and replicated four times. Results showed that enzyme inclusion had a dramatic effect on gut viscosity but there was little difference in response between the two levels of enzyme. There was also an increase in digestible energy measured in the hindgut with feed enzyme addition and sometimes in feed conversion ratio (FCR). In vitro viscosity had a good correlation (R2=0.53) with liveweight gain; this was particularly strong after viscosity exceeded 2.5 cps. However, when the in vitro viscosity of the cereal grains was related to excreta stickiness score, there was a good relationship (R2=0.82). Similarly in vitro viscosity correlated well with feed intake (R2=0.70) ileal digestible energy (R2=0.73) and in vivo viscosity (R2 =0.73). Implications The need to screen extensively for low-energy wheats using conventional methods is time consuming and impractical, particularly as only very few would be

considered to be of low AME and therefore of high viscosity. Many measurements were made on a large number of samples to examine possible predictive measures. The in vitro viscosity of wheats but not of diets generally were closely correlated with liveweight gain. Although this was a satisfactory outcome, the methods of extracting viscous material are time consuming. In order to provide industry with a rapid and simple test to predict low AME wheats, a method based on excreta moisture or stickiness score might be the most useful. But this needs to be tested and then correlated with gut viscosity and bird performance with and without a feed enzyme. Publications Petersen, S.T. and Farrell, D.J. (1996) Viscous grains - in

the thick of it. Proc. Qld. Poult. Sci. Symp. 5: 14.1-14.8.

Petersen, S.T. and Farrell, D.J. (1996) A comparison of cereal and grain legume diets on several digestive parameters in broilers. Proc. 1997 Aust. Poult. Sci. Symp. 9: 149-152.

Project Title Determination of true amino acid digestibility of feedstuffs RIRDC Project No: US-67CM Researcher: A/Prof Wayne Bryden and Dr

Ravi Ravindran Organisation: University of Sydney Dept of Animal Science CAMDEN NSW 3570 Contacts: Phone: (02) 4655 0658 Fax: (02) 4655 0693 E-mail: [email protected] Objectives • To determine the true amino acid availability of a

range of feed ingredients using guanidinated protein and use these values in feed formulation.

• To predict the magnitude of endogenous amino acid loss from dietary composition.

Background The project was initiated in response to the need in the poultry industry to develop a better feed formulation system than one based on total amino acids. Amino acid digestibility is not the same for all feed ingredients, with some having lower digestibility than others. The use of digestible amino acid values in feed formulations should therefore more consistently meet the birds’ requirements and improve the efficiency of feed utilisation than the currently employed total amino acid system. However, the range of Australian feed ingredients for which digestible amino acid values are available is limited. This project was therefore directed towards developing a database of digestible amino acid values for a wide range of Australian feed ingredients.




Research Ileal amino acid digestibilities, both true and apparent, in a range of Australian feedstuffs were generated in a series of digestibility assays. The homoarginine marker was used to distinguish between exogenous and endogenous amino acids, and to determine true digestibility values. A number of studies were undertaken to assess the validity of the basic assumptions underlying the use of this homoarginine technique. While the determination of true ileal amino acid digestibility values (using the homoarginine technique) focused on selected ingredients, apparent ileal digestibility values were determined for a large number of samples with the object of characterising the variability within locally available ingredients. Outcomes Comparisons of digestibility estimates at ileal and excreta levels demonstrated that analysis of digesta from the terminal ileum rather than excreta will yield more accurate values of amino acid digestibility in feed ingredients for poultry. A protocol for the determination of endogenous amino acid output under continuous feeding conditions using the homoarginine technique was developed. True digestible amino acid values for four samples of soybean meal, two samples of cottonseed meal, sunflower meal, meat and bone meal, canola meal, gelatine and three samples of casein were determined. Apparent ileal digestibility values were determined for over 72 samples representing 22 different feed ingredients. A database was compiled of the apparent ileal amino acid digestibilities of Australian feed ingredients for broilers. This database was published by RIRDC and made available to the industry in February 1998. Studies undertaken also showed that both apparent and true digestibility values are additive and that the digestible amino acid supply in a complete diet can be predicted, with reasonable accuracy, based on amino acid digestibility determined for individual feed ingredients. Implications The database of digestible amino acid values generated by this project will enable industry nutritionists to fine-tune their feed formulations, thereby lowering feed costs and improving the feed efficiency of broiler production. Publications Angkanaporn, K., Ravindran, V. and Bryden W.L .(1996)

De novo synthesis of homoarginine in chickens is influenced by dietary lysine and arginine. Nut. Res.17: 99-110.

Angkanaporn, K., Ravindran, V. and Bryden W.L. (1996) The measurement of endogenous amino acids in the excreta of adult cockerels. Archiv. fur Geflugel-Kunde. 60: 260-267.

Angkanaporn, K., Ravindran, V. and Bryden W.L. (1996) Additivity of apparent and true ileal amino acid digestibilities in soybean meal, sunflower meal,

and meat and bone meal for broilers. Poult. Sci. 75: 270-276.

Angkanaporn, K., Ravindran, V. and Bryden W.L. (1997) Evaluation of homoarginine as a marker for the determination of endogenous amino acid concentrations in excreta of poultry. Brit. Poult. Sci. 38: 577-585.

Angkanaporn, K., Ravindran, V. and Bryden W.L. (1997) Homoarginine influences voluntary feed intake, tissue basic amino acid concentrations and kidney arginase activity in broilers. J. Nut. 127: 1128-1136.

Angkanaporn, K., Ravindran, V. and Bryden W.L. (1997) Secretion of homoarginine into the gut of chickens. Vet. Res. Communications 21: 161-167.

Ravindran, V., Hew, L.I. and Bryden, W.L. (1998) Digestible amino acids in poultry feedstuffs. RIRDC Publication No 98/9 US-67CM.

Ravindran, V., Hew, L.I. and Bryden, W.L. (1996) Ileal digestibilities of amino acids in broiler feeds. Proc. Tenth Aust. Poult. and Feed Conv., Melbourne, pp.215-219.

Ravindran, V., Hew, L.I. and Bryden, W.L. (1996) Guanidination of lysine in cottonseed protein. J. Food Ag. Chem. 44: 1812-1815.



3.1 CHICKEN MEAT RESEARCH IN PROGRESS

Project Title Junior Research Fellowship - Ms Lisa Alleva RIRDC Project No: ANU-21A Start Date: 1 July, 1995 Finish Date: 30 June, 1998 Researcher: Dr Karen Ovington and Ms

Lisa Marie Alleva Organisation: Australian National University

Division of Biochemistry & Molecular Biology ACT 0200


E-mail: [email protected] Objectives • To investigate the role that the cytokine TNF-α plays

in host responses to infection with intestinal coccidia belonging to the genus Eimeria.

Current Progress It has been clearly shown that TNF-α has important effects on the host's ability to limit parasite reproduction. As theoretically predicted, this cytokine was shown to be produced at the site of infection. Unexpectedly though, TNF-α produced in the intestine or TNF-α injected into infected animals increased the reproduction of the parasite. A series of experiments using genetically modified mice, TNF-α antagonists, and recombinant TNF-α provide convincing evidence that TNF-α does not directly affect the development of Eimeria but is pro-parasitic because of host responses it induces after binding to one of the two host receptors for TNF-α. TNF-α bound to TNF Receptor I is pro-parasitic, at least partly because it inhibits the host's ability to produce interferon-α which is critical in responses that limit parasite reproduction. Further, TNF-α may stimulate excessive production of reactive oxygen and nitrogen intermediates, potentially damaging host effector cells which limit parasite stages that can establish in the intestine. These results are important to consider for coccidiosis vaccine strategies as they suggest that antigens that stimulate TNF-α production should be excluded from vaccines. Project Title Control of ovarian development and function in meat chicken females RIRDC Project No: CSJ-2A Start Date: 1 January, 1996 Finish Date: 30 June, 1998 Researcher: Dr Jeff Downing Organisation: CSIRO Division of Animal

Production

c/- University of Sydney Dept of Animal Science

Werombi Road CAMDEN NSW 2570 Contacts: Phone: (02) 4655 0600

Fax: (02) 4655 0693 E-mail: [email protected] Objective • To determine the mechanisms by which growth

factors and their associated proteins control the development of ovarian follicles in broiler breeder hens and to develop technologies to improve their reproductive performance.

Current Progress Previous in vitro studies showed that fibroblast growth factor (FGF) is involved in the development of ovarian follicles and its action can be inhibited by hexadimethrine (HDM). In the past 12 months in vitro work has been directed towards isolating FGF from granulosa cell conditioned media and studying whether this FGF has mitogenic activity on ovarian granulosa and thecal cells. These studies have shown endogenous FGF to display activity in both cell types. In vivo studies have been directed at determining the suitability of using HDM to inhibit ovarian follicle development. A radioactive-labelled HDM formulation has been used to show that HDM can be administered in various ways and is taken up by the cells of the ovarian follicle. Following this success studies were undertaken to, firstly, access the effects of HDM on hen health and, secondly, to determine the dose of HDM needed to influence the number of follicles developing in the ovary. Preliminary studies have indicated that HDM can inhibit the number of ovarian follicles that develop but at this stage the doses used have inhibited too many follicles from growing. More extensive work will be needed to determine the suitability of using HDM to influence broiler breeder reproductive performance. Project Title On farm comparison of computer controlled tunnel ventilated and naturally ventilated broiler sheds RIRDC Project No: DAN-40CM Start Date: 1 January, 1995 Finish Date: 30 June, 1999 Researcher: Mr Gerry Bolla Organisation: NSW Agriculture

PO Box 581 GOSFORD NSW 2250






Objective • To compare the performance of two commercial

broiler shed designs viz. (i) computer controlled, insulated, curtain sided tunnel ventilated sheds and (ii) computer controlled, curtain sided, insulated, naturally ventilated sheds. These designs are to be compared on the basis of financial return per square foot of shed space, bird feed conversion ratio, batch mortality rates, bird behaviour and several shed climatic factors (temperature, humidity vs ambient temperature/humidity and daily variations of both).

Current Progress Four sheds (two naturally ventilated and two tunnel ventilated) have been commissioned and computer controllers installed. Data loggers have been installed in each shed to record ambient and shed temperature variations. Substantial modifications of and adjustments to the equipment were made in June 1997 to improve the performance of the sheds and data recording. Overall performance has improved since these modifications were made. To date seven batches of chickens have been used in the trial. With only these seven batches analysed, there are few significant differences between the tunnel ventilated sheds and the naturally ventilated sheds at this stage. Performance can be related to day old chicken quality. At this stage mortality seems to be lower in the tunnel ventilated sheds as does feed conversion ratio. Power consumption is not significantly different between sheds. Return per bird is slightly better in the tunnel ventilated sheds. However both tunnel and natural sheds are performing better than the pool average. It appears the tunnel sheds are easier to manage. Project Title Production of distance learning materials for the poultry industry - stage III continued RIRDC Project No: DAN-142A Start Date: 1 July, 1996 Finish Date: 30 June, 1998 Researcher: Mr Geoff Creek Organisation: NSW Agriculture

Murrumbidgee College of Agriculture PMB

YANCO NSW 2703 Contacts: Phone: (02) 6953 0299

Fax: (02) 6953 0268 Objectives • To produce distance education material suitable for

managers of chicken meat enterprises. • To evaluate these distance learning materials. • To arrange accreditation for the distance education

course “Commercial Meat Chicken Management”.

Current Progress The distance learning materials for this project are being produced as a series of nine units. A number of writers, including NSW Agriculture staff and external consultants, are involved. Unit C (Health and Welfare) was published in 1996. Unit B (Land Use, Housing and Equipment) is in the final stages of refereeing. Unit E (Brooding and Rearing Meat Chickens) is ready for printing. Unit D (Feeding Practices) is at the pre-print stage. Units A (Hatchery and Breeder Management) and F (Running the Business I) are 90% written, and Units G (Running the Business II), H (Industry Issues) and I (Products and Marketing) are approximately 50% written. A matching of the materials to the latest National Competency Standards has identified a need for a complementary project to produce a "Learner's Guide" which will enable managers to be accredited with appropriate competencies. Project Title Infectious proventriculitis and stunting syndrome of broiler chickens RIRDC Project No: DAN-158A Start Date: 15 June, 1997 Finish Date: 30 June, 1998 Researcher: Dr Rod Reece Organisation: NSW Agriculture

Regional Veterinary Laboratory Elizabeth Macarthur Agricultural Institute PMB 8

CAMDEN NSW 2570 Contacts: Phone: (02) 4640 6309

Fax: (02) 4640 6400 E-mail: [email protected] Objective • To control outbreaks of infectious proventriculitis

and stunting syndrome associated with reduced growth and poor performance of broiler chickens by understanding the pathogenesis of the condition, determining its aetiology, developing diagnostic tests and producing an appropriate vaccine.

Current Progress Formalin fixed material for histological examination, and chilled or frozen proventriculi and intestinal tracts for possible transmission trials, have been obtained from several integrated broiler companies from NSW and other States. This material originated from broiler flocks with impaired performance and gross proventricular changes. A lymphoplasmacytic infiltrate into the proventricular lobules, atrophy of the alveoli and proliferation of secondary and tertiary ducts, typical of transmissible proventriculitis as described in the USA, UK and Australia, were found. Interestingly, archival histological material from Victoria has revealed similar lesions in stunted chickens with proventriculitis dated 1982.




Three transmission trials were conducted in collaboration with Steggles Limited using their chickens and isolators. Reduced growth rate compared to controls and typical gross and histological lesions were observed in broiler and specific pathogen free (SPF) chickens inoculated with intestinal and proventricular homogenates. Lesions were detected at five days post inoculation. The SPF chickens seroconverted to chicken anaemia virus (CAV) and a few died showing anaemia but it is not certain at this stage if CAV was in the original inoculum or was an environmental contaminant. Further trials are planned to answer this question using earlier material. A proventricular disease resembling transmissible viral proventriculitis has been found to be transmissible using crude homogenates and was also passageable by using homogenates of intestines and proventriculi from inoculated chickens. The homogenates contained a range of potential pathogens, the significance of each of which is unknown in the full manifestation of the disease. Inoculated chickens have been consistently seronegative for reticuloendotheliosis virus. The incidence of proventriculitis in inoculated chickens has varied from 10-80%. Histology from the last trials has not yet been finalised. Further transmission trials are planned. Project Title The effect of Salmonella Sofia on intestinal infection with other salmonellae in chickens RIRDC Project No: DAV-120A Start Date: 1 July, 1996 Finish Date: 30 June, 1998 Researcher: Dr Colin Wilks Organisation: Dept of Natural Resources &

Environment Victorian Institute of Animal Science

475-485 Mickleham Road ATTWOOD VIC 3049 Contacts: Phone: (03) 9217 4200

Fax: (03) 9217 4299 E-mail: [email protected] Objective • To test the hypothesis that intestinal colonisation in

chickens with Salmonella enterica subspecies II serovar Sofia, limits colonisation with salmonellae that may be pathogenic for humans and/or chickens, and that this effect is mediated by (a) competitive exclusion or (b) intestinal immune mechanisms.

Current Progress In the initial stage of this project clones of Salmonella Sofia and Typhimurium were produced that were resistant to a specific antibacterial (nalidixic acid) so that direct isolation and enumeration of organisms from the faeces of infected birds could be performed. This was followed by determining the dose, timing and route of exposure required to reproducibly produce colonisation in chickens with these Salmonella strains. It was found that chickens could be infected with Sofia by either

spraying eggs one day prior to hatch or by oral inoculation of chicks at one day of age. Infection with Typhimurium was readily established by oral inoculation at five days of age. A practical model was therefore established for assessing the protective effect of prior colonisation with Sofia on subsequent challenge with Typhimurium. When Sofia-colonised birds were challenged with Typhimurium, however, no protective effect was observed and all challenged birds became persistently colonised with Typhimurium which, in turn, displaced the Sofia. The colonising ability of several isolates of Sofia has subsequently been evaluated and significant differences between isolates identified. It is planned that these protection experiments be repeated to determine whether or not one of these other strains of Sofia is a better coloniser and therefore more able to block colonisation with Typhimurium. Project Title The control of big liver and spleen disease (BLS) RIRDC Project No: DAW-74A Start Date: 1 July, 1996 Finish Date: 30 June, 1999 Researcher: Dr Trevor Ellis Organisation: Agriculture Western Australia

Animal Health Laboratories Locked Bag No 4 BENTLEY DELIVERY CENTRE WA 6983


E-mail: [email protected] Objective • To reduce the financial losses associated with BLS

by the development of appropriate diagnostic and control measures, including vaccination schedules.

Current Progress Strain variation studies on BLS isolates have been completed and have shown no detectable variation between strains by virus neutralisation, ELISA or Western blotting techniques. Serum from SPF (specific pathogen free) birds infected with strain BLS 86 neutralised all five distinct BLS isolates. ELISA antigens from all viruses were tested against polyclonal and monoclonal antibody panels and all gave equivalent titres against the five viruses. By Western blotting, using monoclonal or polyclonal antibodies, no differences were found between isolates. Monoclonal antibody (MAb) panel development work has produced six extra specific BLS MAbs that were used with existing MAbs for strain variation studies. Serum from birds vaccinated with a 16-18 kDa BLS protein neutralised all five BLS strains. Pen trials to evaluate the vaccine potential of this protein are in progress, using the optimum vaccine/adjuvant combination previously determined. The first group of birds tested were found to have pre-existing BLS infection in some vaccinates and controls so the study has to be repeated. However, challenge of the groups did





show that vaccinated birds cleared virus from liver and spleen significantly more rapidly (P < 0.006) than controls. Protein sequencing of fragments of the 16-18 kDa BLS protein has led to the development of putative BLS specific PCR primers. These are being evaluated for development of a PCR for BLS and for potential preparation of recombinant DNA produced vaccine antigen. Project Title Molecular basis of benign colonisation of Salmonella Sofia in chickens RIRDC Project No: IMVS-1A Start Date: 1 August, 1997 Finish Date: 30 July, 2000 Researcher: Dr Michael Heuzenroeder Organisation: Institute of Medical &

Veterinary Science Infectious Diseases Laboratory

PO Box 14 Rundle Mall ADELAIDE SA 5000


E-mail: [email protected] Objectives • The conventional and molecular typing service

developed under previous projects will continue to be offered to the industry.

• Salmonella Sofia strains that have been characterised genetically (ie rationally chosen) will be gauged for their ability to colonise and competitively exclude virulent serovars eg. Typhimurium.

• The bacterial factor (fimbriae) involved in the efficient coloniation of chickens by S. Sofia will be identified and genetically characterised by DNA sequence analysis.

• A longer term aim of the research (outside of the time frame of the current project) will be to place the factor characterised in this project into a non-Salmonella organism for competitive exclusion purposes.

Current Progress In 1997/98, no significant change in the Salmonella serovar distribution in Australian meat chickens has occurred. Salmonella Sofia is still the most commonly isolated Salmonella serovar from chickens, but is rarely seen in humans. To date, 2668 chicken isolates have been received. Fimbriae are bacterial appendages important for colonisation of host animals. PCR and DNA sequence analysis was used to determine whether the following Salmonella fimbrial genes are present in S. Sofia: lpfA, agfA, sefA, sefD and pefA. The agfA gene was encoded in every strain tested. In contrast pefA was not found.

The lpfA sequence was found in the majority of isolates. This is most unusual since lpfA is not predicted to exist in subspecies II Salmonella, such as Sofia. Similarly sefA and sefD sequence has been found in some but not all isolates. This suggests that S. Sofia has acquired these genes through genetic exchange with other organisms. Studies on the mutagenesis and expression of the agfA and lpfA genes are in progress. Experiments conducted in collaboration with the Victorian Institute of Animal Science have indicated that S. Sofia isolates chosen on the basis of their genetic background and given at low dosages do not persist in chickens. Studies at higher dosages are in progress. Project Title Use of avirulent Campylobacter jejuni strains to control poultry-derived Campylobacter food poisoning RIRDC Project No: RMI-7A Start Date: 1 July, 1997 Finish Date: 30 June, 2000 Researcher: Dr Victoria Korolik and Dr

Peter Coloe Organisation: Royal Melbourne Institute of

Technology GPO Box 2476V

MELBOURNE VIC 3001 Contacts: Phone: (03) 9660 2110

Fax: (03) 9662 3421 E-mail: [email protected] Objectives • To detect and identify those strains of

Campylobacter jejuni that colonise chickens but do not constitute a disease problem in humans.

• To establish a typing scheme for Campylobacter spp. that will be available to characterise flock isolates.

• To develop a molecular typing scheme that will distinguish human virulent strains of Campylobacter spp. from normal flora animal strains.

• To develop an inoculation mixture for use in chickens that will constitute avirulent ‘competitive exclusion’ strains of Campylobacter spp. for use in chickens.

Current Progress Thermophilic Campylobacter species such as C. jejuni and C. coli are recognised as major causes of acute gastroenteritis world-wide in man through consumption of contaminated foods, including poultry. One of the major objectives of this project was to develop a PCR-based test that can differentiate strains of C. jejuni isolated from humans and from chickens. The test is based on the DNA sequence polymorphism of a ClaI restriction enzyme site on the C. jejuni genome based on the DNA probe pMO2005 constructed previously. The DNA sequence of the C. jejuni DNA fragment containing the polymorphic ClaI site (total of 1800 bp) has been





determined and PCR primers have been designed for differentiation of the majority of C. jejuni strains. PCR analysis using these primers resulted in amplification of ~700bp DNA fragment in all C. jejuni strains tested. The amplified DNA fragment could be cleaved using ClaI to produce a two band pattern in strains carrying the polymorphism. The DNA from other Campylobacter spp. was not amplified using the same PCR test. Such a test could be used to quickly and accurately screen chicken flocks for the presence of C. jejuni and to determine to which majority group the strains belong. Project Title Managing structural change in the Australian poultry industry RIRDC Project No: SGH-5A Start Date: 15 February, 1998 Finish Date: 15 December, 1998 Researcher: Mr Terry Larkin Organisation: S.G. Heilbron Consulting Pty

Ltd c/- 46 Creswell Street CAMPBELL ACT 2612


E-mail: [email protected] Objective • To provide the Australian chicken meat industry

with a framework and pathways to manage the major structural changes it is facing from internationalisation.

Current Progress Preliminary research and a benchmarking review has been completed. Discussions with industry and officials is continuing. An industry survey has been designed and questionnaires for the first round of new data collection have been despatched to processors and growers. A good survey response and a high level of industry and government cooperation in the next phases of the project will be important. This is an innovative project in an industry which has been characterised by adversarial relationships among stakeholders, both public and private, and further progress will be dependant on a high degree of stakeholder cooperation. Project Title Identification of in vivo expressed antigens of Pasteurella multocida RIRDC Project No: UMO-12CM Start Date: 1 January, 1995 Finish Date: 30 June, 1998 Researcher: Dr Ben Adler Organisation: Monash University

Dept of Microbiology CLAYTON VIC 3168


E-mail: [email protected] Objectives • To identify genes encoding proteins (antigens)

whose expression is switched on during growth in vivo.

• To clone and sequence analysis of the genes so identified.

• To replace the regulated promoters of these genes with unregulated, strong promoters, allowing constitutive expression of these proteins.

• To immunise mice and SPF chickens with these antigens in order to assess their ability to cross-protect against P. multocida infection.

Current Progress A genetic system was established to identify genes of P. multocida, the causative agent of fowl cholera, which are expressed during the growth of the bacteria during infection in vivo (ie. within the animal). The system was used to identify a number of genes which, together with others identified in the laboratory, are being assessed for their potential as either the basis for attenuating mutations to construct live vaccines and/or for the potential of the encoded proteins as vaccine antigens. Genes which have been identified include those encoding surface iron-binding proteins, fimbriae involved in adhesion to host surfaces, a haemolytic esterase, other haemolysins, a zinc finger regulatory protein, the surface capsule, an ABC surface transporter protein and a hitherto unknown surface protein. At least a part of each of these genes has been cloned and sequenced. For four of them, the whole gene has been obtained and cloned into an expression vector to overexpress the protein. One mutant strain has been constructed and three more are currently under construction. In future work, mutants and expressed proteins will be assessed for their vaccine potential in chickens. Project Title The "new season grains" phenomenon and the role of the endogenous glycanases in grains for their nutritive value in poultry RIRDC Project No: UNE-53A Start Date: 1 August, 1996 Finish Date: 1 August, 1999 Researcher: Dr Mingan Choct Organisation: University of New England

School of Rural Science and Natural Resources – Animal Science

ARMIDALE NSW 2351 Contacts: Phone: (02) 6773 5121

Fax: (02) 6773 3275 E-mail: [email protected]






Objective • To investigate the "new season grains" phenomenon,

in particular the role of endogenous glycanases (non-starch polysaccharide-degrading enzymes) in cereal grains for their nutritive value in broiler chickens.

Current Progress Four samples of the cereal grains, wheat, barley, triticale and oats, were collected at the time of harvest and their apparent metabolisable energy (AME) values were determined as a measure of their nutritive value for broiler chickens. AME trials were conducted at approximately three-month intervals, with the first one being completed within 30 days of harvest. The results from the 1996/1997 harvest season demonstrated that the post-harvest changes in AME of cereal grains differed widely, with wheats responding most positively (10-25% increase in AME) to storage. Barleys and oats also responded positively (0-6% increase in AME with storage), but the magnitude of increase was smaller compared to wheats. The AME of triticale, sorghum and maize did not change with storage. Most of the increase in AME occurred after three months of storage. The endogenous enzyme activities in these cereal grains were also measured using an extract viscosity method. The level of arabinoxylanase activity was high in oats, whereas wheat and triticale had significant β-glucanase activity. It is possible that the structure of the grain cell wall undergoes changes during storage due to endogenous enzyme activities and these changes are favourable for digestion of the nutrients contained in the grain. It appears that the post-harvest change in the nutritive value, in particular the AME values, of wheat and barley for poultry is significant and has important practical implications for the feed and livestock industries. Project Title Postgraduate scholarship - Andreas Kocher: Increasing the nutritive value of grain legumes for poultry by use of more efficacious enzyme systems RIRDC Project No: UNE-64A Start Date: 1 July, 1997 Finish Date: 30 June, 2000 Researcher: Dr Mingan Choct and Mr

Andreas Kocher Organisation: University of New England

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


E-mail: [email protected]

Objective • To investigate the effect of commercial feed

enzymes on the nutritive value of grain legumes. Current Progress The effect of three commercial enzyme products on the nutritive value of two types of lupin, L. angustifolius cv Gungurru and L. albus cv. Kiev mutant, were determined in a classical AME (apparent metabolisable energy) bioassay. In a factorial design, 12 experimental diets (three types of lupin: no lupin, Gungurru, Kiev; four types of enzymes: no enzyme, enzyme 1, enzyme 2, enzyme 3) were tested. Digesta from duodenum, jejunum, ileum and caeca were collected to determine the digestibility of protein and non-starch polysaccharide (NSP). The results from the AME study showed that addition of commercial enzyme products to lupin based diets did not have a significant effect on performance (growth and feed conversion ratio) or AME. However, the addition of an enzyme complex derived from the fungus Aspergillus aculeatus to Gungurru based diets resulted in a significant increase in digesta viscosity in all sections of the intestine. The increase in viscosity is the result of increased soluble NSP. It is assumed that the pectinase in the enzyme complex attacked the bond of the polysaccharides, which prevented the NSP from dissolving. Most of the increase in soluble NSP was galactose and arabinose, which shows that the enzyme most likely attacked parts of the galactans and arabinan present in lupins. Although not significant, it appears that the addition of an enzyme complex derived from Trichoderma viride to Gungurru based diets also solubilised parts of the insoluble NSP. There were no significant differences between the Kiev basal diet and the three diets supplemented with enzymes. This clearly highlights the differences in the structure of NSP between the two lupin species. Project Title Defined probiotic preparations for competitive exclusion of enteropathogens from poultry RIRDC Project No: UNS-10A Start Date: 1 March, 1997 Finish Date: 31 March, 2000 Researcher: Dr Julian Cox Organisation: University of New South

Wales Dept of Food Science and Technology

SYDNEY NSW 2052 Contacts: Phone: (02) 9385 5665






Objective • To develop fully defined microbial consortia which

prevent colonisation of meat chickens with enteric pathogens, particularly Salmonella, thereby markedly reducing the significance of poultry as a vehicle of transmission for these pathogens.

Current Progress A continuous, anaerobic culture was initiated using caecal material from a Salmonella-free adult broiler chicken. This culture has been maintained for over 12 months. Enumeration and observation of the microflora was conducted after 24 hours, seven days and two months. A substantial qualitative change in the microflora was observed between these three sampling events, and a considerable biofilm formed in the fermenter after approximately four weeks. Further qualitative evaluation has shown that the microflora has remained essentially stable since that time. Preliminary characterisation of strains showed that most are Gram-positive, facultatively anaerobic rods and cocci. Experiments have been conducted to examine the in vitro sensitivity of pathogens to the anaerobic culture. Several strains of Salmonella are rapidly eliminated (within 24 hours), even when inoculated into aliquots of the anaerobic culture at 106 cfu/ml. Interestingly, a strain of Salmonella Enteritidis, induced to produce hydrophobic SEF17 fimbriae, was more resistant, but was still eliminated within 48 hours. Similarly, Salmonella Sofia was eliminated more slowly. In vivo experiments, using day-old chicks in a standard competitive exclusion assay, are about to commence. Project Title Odour and ammonia emission reduction from chicken broiler growout farms RIRDC Project No: UNS-11A Start Date: 1 September, 1996 Finish Date: 30 August, 1998 Researcher: Mr John Jiang Organisation: Uni. of New South Wales

Centre for Wastewater Treatment School of Civil Engineering


Fax: (02) 9313 8624 E-mail: [email protected] Objectives • To quantify odour and ammonia emissions in and

adjacent to some typical meat chicken farms and to identify and make recommendations on potential remedial measures with a particular focus on operational and management aspects.

• To apply odour emission data generated by the project to the Auspuff and Ausplume dispersion models.

• To undertake additional odour sampling on four typical Victorian chicken broiler farms.

Current Progress Field work and data analysis for a full year emission study at two NSW farms and spot surveys on eight other farms in Victoria and NSW have been completed. A modern, forced choice dynamic olfactometer was used to determine odour concentration (NVN 2820) and odour intensity (VDI 3882). Ammonia concentration, litter characteristics and environmental parameters such as temperature, humidity and wind speed were also recorded. The study has covered naturally ventilated, cross flow and tunnel ventilated sheds, a range of litter types and a range of climatic conditions in south eastern Australia. Data obtained showed that the peak odour concentration inside a shed on a broiler growout farm occurred at about week six of the growth cycle, when the total bird biomass in the shed reached a maximum. Wide variations in odour concentrations were found across different types of broiler sheds in NSW and Victoria. Intensity studies indicated that at an odour concentration of 10 OU/m3, panelists perceived the chicken odour as distinct but with further dilution it was no longer perceived as noticeably odorous. Work to apply odour emission data generated by the project to the AUSPLUME and AUSPUFF atmospheric dispersion models is in progress. Project Title Development of a new fowl pox vaccine strain RIRDC Project No: UQ-57A Start Date: 1 May, 1996 Finish Date: 30 April, 1999 Researcher: Prof Wayne Robinson Organisation: University of Queensland Dept of Veterinary Pathology

ST LUCIA QLD 4072 Contacts: Phone: (07) 3365 2565

Fax: (07) 3365 1355 E-mail: [email protected] Objectives • To develop a candidate fowl pox vaccine strain that

is of greater immunogenicity than the existing strain. • To identify the factor or factors that have led to

recent outbreaks of fowl pox in the face of vaccination.

Current Progress In the quest for the development of a new vaccine against fowl pox virus, the following progress has been made. A wide variety of field isolates were obtained from a number of Australian States, especially NSW. All have been tested for the presence of reticuloendotheliosis virus (REV) and all except two fowl pox isolates from wild birds were found to contain REV. Given that all field strains contained REV, isolates from Queensland, NSW and Victoria were assessed for their ability to protect against challenge. In laboratory trials, an isolate from





Victoria and an isolate from NSW have shown encouraging results with respect to protection against challenge. At present, a larger laboratory trial is underway. It is possible that a vaccine can be developed from one of these two field isolates. Project Title Development of DNA vaccines and techniques for cytokine manipulation for improved mucosal immunity in chickens RIRDC Project No: US-34A Start Date: 1 January, 1996 Finish Date: 31 March, 1999 Researcher: Prof Alan Husband and

Dr Wendy Muir Organisation: University of Sydney

Dept of Veterinary Anatomy and Pathology


Fax: (02) 9552 3266 E-mail: [email protected] Objectives • To extend work already undertaken towards novel

mucosal vaccines by developing mucosal applications for nucelotide (DNA) vaccines in association with local gene therapy for expression of biological response modifiers (cytokines).

• To continue investigations of the basic mechanisms of mucosal immunity in chickens.

Current Progress Recent research work has focussed on identifying avian cytokines, which function as messengers and regulators of the immune system, that are involved in regulating mucosal, IgA predominant, intestinal immune responses in chickens. Based on the limited knowledge of avian cytokines and the function of their mammalian counterparts, chicken myelomonocytic growth factor (cMGF) and transforming growth factor beta (TGF-β) have been selected as potentially important cytokines. Their involvement in intestinal immunity has been assessed by in vitro and in vivo studies. Within two hours of in vitro stimulation of whole chicken blood with lipopolysaccharide or tetanus toxoid the expression of both of these cytokines was identified by reverse transcription polymerase chain reaction (RT-PCR). Following vaccination of chickens for the specific induction of intestinal IgA antibody, samples of blood and tissues, in particular caecal tonsils, duodenum and spleen, have been analysed for these two cytokines. Under these conditions expression of cMGF is rare but it is hoped that in vitro stimulation of blood taken from these chickens should facilitate the detection of low levels of cMGF expression. TGF-β is produced by all chickens, irrespective of their immunisation status. However, it has been confirmed that higher levels of TGF-β are expressed in immunised chickens.

Project Title The development of a model of abnormal bone development in broiler chicken as an aid in the assessment of welfare RIRDC Project No: US-35A Start Date: 1 May, 1996 Finish Date: 1 August, 1999 Researcher: A/Prof Wayne Bryden Organisation: University of Sydney

Dept of Animal Science Werombi Road


Fax: (02) 4655 0693 E-mail: [email protected] Objectives • To develop a model of tibial dyschondroplasia (TD)

using the mycotoxin fusachromanone. • To examine the influence of factors reported to

modulate the incidence of disease in this model. Current Progress Fusachromanone is a mycotoxin that induces lesions in birds indistinguishable from tibial dyschondroplasia. The initial studies of the project have been directed towards developing a model for the development of this skeletal abnormality using this mycotoxin. Fungal isolates that produce the toxin have been obtained from both the United States and Denmark and used in laboratory studies to produce toxin. Feeding studies with broiler chickens have been successful in using this toxin to reproduce this syndrome. A most important observation, which has been repeated in our studies, demonstrates that there are significant differences between broiler strains in their susceptibility to induction of tibial dyschondroplasia by the toxin. Interestingly, the differences in susceptibility in this model correspond to the differences in susceptibility in field studies. Project Title Broiler feed formulation based on digestible amino acids RIRDC Project No: US-38A Start Date: 1 January, 1997 Finish Date: 30 June, 1998 Researcher: A/Prof Wayne Bryden and Dr

Ravi Ravindran Organisation: University of Sydney









Objectives • To evaluate the formulation of broiler diets on a total

amino acid basis vs digestible amino acid basis, with particular reference to inclusion of cheaper, alternative feed ingredients.

• To examine the utility of the digestible amino acid concept in low-protein diets for broilers.

Current Progress Digestible amino acid (AA) values are likely to form the basis of poultry feed formulations in the future. Despite this, currently few commercial nutritionists formulate diets based solely on digestible AAs, largely because limited published information is available on broiler responses to diets formulated on the basis of digestible AAs. In a series of broiler growth assays, diets containing graded levels of canola meal, cottonseed meal or meat and bone meal and formulated on the basis of total or digestible AA concentrations of the ingredients, were first determined and then diets containing graded levels of these ingredients, formulated on the basis of total vs digestible AA and fed to broilers in short-term feeding trials. The results demonstrated that differences in digestible AA content will result in comparable differences in broiler performance and that formulating broiler diets on a digestible AA basis will allow poorer quality ingredients to be incorporated at higher levels without adversely affecting bird performance. It appears that the lower the AA digestibility of an ingredient, the greater will be the benefit of employing digestible AA values in feed formulations. Project Title Manipulation of lean tissue deposition by altering the sensitivity of tissues to insulin RIRDC Project No: US-44A Start Date: 1 January, 1997 Finish Date: 31 December, 1999 Researcher: A/Prof Wayne Bryden and

Mr Ron Newman Organisation: University of Sydney



Fax: (02) 4655 0693 E-mail: [email protected] Objective • To reduce fat deposition and increase muscle protein

synthesis in the broiler by altering the sensitivity of tissues to the metabolic hormones in lipid and protein metabolism.

Current Progress Changes in body composition can be achieved by modulating the sensitivity of tissue to the metabolic hormones that control lipid and protein metabolism. To

measure these changes, a series of techniques have been developed. A radioimmunoassay specific for the measurement of chicken insulin has been established. Two different hyperglycaemic and euglycaemic infusion techniques for the in vivo measurement of insulin sensitivity have also been established. The surgical procedures required for the above infusion techniques and for repetitive blood sampling in chickens have also been developed. A chicken muscle cell culture system for the in vitro measurement of insulin sensitivity has been created, however experiments to measure insulin sensitivity using the method are yet to be carried out. An initial experiment to determine if dietary fats can alter carcass composition and impart measurable physiological changes in broiler chickens has been completed. The results from this experiment suggest that dietary long chain polyunsaturated fatty acids can reduce fat deposition without impairment of growth rate. Experiments to determine the differences in growth performance and metabolism between broiler and layer strains are underway. Project Title Expression of Eimeria genes in Toxoplasma RIRDC Project No: UTS-1A Start Date: 1 December, 1995 Finish Date: 15 June, 1999 Researcher: Dr John Ellis Organisation: University of Technology,

Sydney Dept of Cell and Molecular Biology PO Box 123

BROADWAY NSW 2007 Contacts: Phone: (02) 9330 4161

Fax: (02) 9330 4003 E-mail: [email protected] Objectives • To express Eimeria antigen genes in Toxoplasma. • To investigate the use of novel recombinant

Toxoplasma parasites as a live vector vaccine against poultry coccidiosis.

Current Progress The genetics and molecular biology of the parasite Toxoplasma gondii have been extensively studied in recent years. These advances have been aided by the development of transfection systems that allow the introduction, into T. gondii, of foreign DNA. This technology has therefore many potential applications in parasite research, including the development of novel live vaccines. This project will utilise transfection technology in order to investigate the feasibility of expressing genes from Eimeria tenella in T. gondii and to investigate the vaccine potential of such transgenic Toxoplasma against poultry coccidiosis. Progress to date has focussed on the establishment of transfection technology for T. gondii which is now in place.





Project Title A maternally delivered vaccine against coccidiosis in broiler chickens RIRDC Project No: UTS-2A Start Date: 1 December, 1997 Finish Date: 30 November, 1998 Researcher: Dr Nicholas Smith Organisation: University of Technology,

Sydney Molecular Parasitology Unit Dept of Cell and Molecular Biology PO Box 123

BROADWAY NSW 2007 Contacts: Phone: (02) 9514 4013

Fax: (02) 9514 4026 E-mail: [email protected] Objective • To prove the feasiblity of utilising maternal

immunity in the control of avian coccidiosis by determining whether it is possible to induce the production of maternal antibodies to protect hatchlings against infection with many species of Eimeria by immunisation of breeding hens with a single species of Eimeria.

Current Progress A flock of broiler breeder hens and roosters was established and divided into six groups of 24 hens plus 2 roosters. Egg production and Eimeria oocyst contamination of the litter were monitored every week and the egg yolks analysed for the presence of anti-Eimeria antibodies. Oocyst contamination in the litter proved minimal and the levels of anti-Eimeria antibodies in the egg yolks were not significantly different to those from known uninfected hens. Once egg production reached acceptable levels and fertility was confirmed, the hens were injected intramuscularly with 0µg, 0.2µg, 0.5µg or 2.0µg of purified Eimeria maxima gametocyte antigens emulsified in a commercially acceptable adjuvant. The two remaining treatment groups were either untreated or orally infected with E. maxima (positive control). Eggs are being collected on a weekly basis and analysed for anti-Eimeria antibodies. Groups of eggs will be incubated to hatching and the hatchlings challenged with E. maxima, E. tenella or E. acervulina to determine the level of maternal immunity to these different species resulting from the vaccination regimen. These challenge trials will continue until the end of 1998.




3.1 & 3.2 CHICKEN MEAT & EGG INDUSTRY

COMPLETED JOINT PROJECTS Project Title Infectious bursal disease virus (IBDV): To determine if current vaccination strategies prevent the emergence of very virulent IBDV strains in Australia RIRDC Project No: CSK-3A Researcher: Dr Jagoda Ignjatovic and

Dr Sandra Sapats Organisation: CSIRO Division of Animal

Health Private Bag No 24 GEELONG VIC 3052

Contacts: Phone: (03) 5227 5000 Fax: (03) 5227 5555 E-mail: [email protected] Objectives • To determine if variant IBDV strains are present in

Australia by comparing the VP2 proteins of field isolates to that of other IBDVs and vaccine strains.

• If variant IBDV strains are present, to determine whether existing vaccination protocols provide protection.

Background Clinical IBDV has been well controlled in Australia. However, in late 1994 and early 1995 there were indications that commercial flocks at a number of sites were experiencing problems with IBDV and that these might be caused by variant IBDV strains. The poultry industry was concerned to know whether there were recent changes in the IBDV strains predominating in Australia and, if so, whether there were implications in terms of vaccination strategies applied for the control of endemic strains. Research Research carried out aimed to: • Isolate and characterise IBDV strains from flocks

with clinical IBD. • Determine if IBDV strains in Australia are changing

in either antigenicity or pathogenicity. • Compare local IBDV strains with those of other

countries. • Determine if current vaccines provide protection

against these recent field isolates. Outcomes A number of field strains of IBDV were isolated from commercial sites in two States. Those isolated in Victoria were confirmed to be true antigenic variants by a number of means, including cross-protection studies. Cross-protection experiments using commercial broiler chickens showed that some of the variants could break through a level of maternal antibodies that would be

expected to be protective and at flock age that would result in immunosuppression. Implications The results indicate that, as is the case in other countries, IBDV strains in Australia are changing. They also suggest that, while new vaccines to protect against these variant IBDV strains may not yet be needed, attention should be paid to maintenance of high antibody titres in breeders to minimise the risk of virulent challenge of broilers at an early age. Project Title Testing and improving the nutritional value of grain legumes RIRDC Project No: DAQ-30CM and DAQ-28E Researcher: Mr Paul Mannion,

Dr David J Farrell and Dr Rider A Perez-Maldonado

Organisation: Dept of Primary Industries (Qld) Queensland Poultry Research and Development Centre

PO Box 327 CLEVELAND QLD 4163 Contacts: Phone: (07) 3824 3081

Fax: (07) 3824 4316 E-mail: [email protected] Objectives • To determine the practical inclusions of field peas,

sweet lupins, faba beans and chick peas in the diets of laying hens and broiler chickens using chemical analyses and production experiments. Some methods of improving their nutritive value were also examined.

Background Conventional feed ingredients for intensive livestock are in increasing demand, in part due to competition from Asian countries. There will therefore be a need to examine alternative food sources particularly of protein-rich ingredients. Grain legume seeds are being used in increasing quantities but there is uncertainty as to what their upper level of inclusion should be in broiler starter and finisher diets and in layer diets. Several grain legumes contain anti-nutritional factors which, at certain inclusions, can impair bird performance and/or have an undesirable effect on other characteristics eg. organ weight, excreta stickiness. Research In three experiments with laying hens and three with broilers comparisons were made between the four grain legumes (field peas, sweet lupins, faba beans and chick peas) included in practical diets at different level of inclusion. Detailed chemical analyses identified the different nutritional characteristics of these grain legumes. These related to energy, protein, amino acids, tannins and the non-starch polysaccharides (NSP). A





layer experiment in which each grain legume was included in diets at 250 g/kg, identified faba beans to be undesirable at this inclusion. Egg production and egg size were depressed. Food intake was highest on the sweet lupin-based diet. Measurements made on the liquid fraction of the gut digesta of hens indicated that the diet with sweet lupins gave a high viscosity reading and the hens on chick pea diets had enlarged pancreases. These observations were also seen in broilers on these same grain legumes. The second layer experiment was terminated prematurely (after ten weeks) because of a suspected disease outbreak. Here only sweet lupins and faba beans were examined to determine the effects of steam pelleting, food enzymes and removal of hulls from faba beans. The latter responded to heat treatment and gave higher hen-day production than sweet lupins. This result was contrary to the finding in the first experiment. The third layer experiment, which was similar to experiment two in design, showed no differences in bird performance between the combined sweet lupin diets when compared with the combined faba bean diets. There was a non-significant tendency for egg weight to be lower when faba beans were included in diets. This was significantly so in experiment one. Egg weight was also lower when sweet lupin diets were steam pelleted when compared to cold pelleted. Egg production was substantially higher in this experiment than in experiment two and these data are probably more reliable as the experiment ran for 16 weeks and birds were in good health. The three broiler experiments were aimed at establishing maximum and industry acceptable dietary inclusion levels of the four grain legumes and these were tested at 120-360 g/kg. Broilers during the finisher stage appeared to be more tolerant of very high levels of the grain legumes than broilers during the starter phase where chick peas and sweet lupins were inferior to field peas and faba beans using growth rate and FCR to 21 days. Excreta stickiness score was much higher on the sweet lupin-based diets which agreed with digesta viscosity measurements. In both starter and finisher phases steam pelleting gave a small but significant increase in growth rate and also in FCR to 21 days. The final experiment was conducted on a semi-commercial scale and identified the upper practical inclusions of the four grain legumes in broiler finisher diets. Unexpectedly, chick peas and field peas did not give as good performance as sweet lupins and faba beans when included at different levels in steam-pelleted diets. Outcomes The results of these experiments identified some problems with sweet lupins and chick peas in that gut viscosity and sticky excreta were characteristic of the former and enlarged pancreases of the latter. For broilers, the recommended upper level of inclusion (g/kg) in diets was, for field peas 300, faba beans 200, chick peas 100 and sweet lupins 100 in both starter and finisher diets, with some reservations regarding lupins. For laying hens, recommended inclusions were 250 g/kg, except for faba beans which was 100-150 g/kg.

Implications These studies provide the feed formulator with important information regarding chemical analyses, anti nutritional factors and safe upper levels of inclusion of these four cultivars of grain legumes. Steam pelleting is beneficial in diets for broilers and possibly in some layer diets. Publications Perez-Maldonado, R.A., Mannion, P.F. and Farrell, D.J.

(1997) The effect of steam and cold pelleting of four grain legumes in broiler starter diets. Proc. 1997 Aust. Poult. Sci. Symp. 9: 133-137.

Perez-Maldonado, R.A., Mannion, P.F. and Farrell, D.J. (1997) The effect of steam and cold pelleting of four grain legumes in broiler finisher diets. Qld. Poult. Sci. Symp. 6: 17.1-17.10.

Perez-Maldonado, R.A., Mannion, P.F. and Farrell, D.J. (1997) Practical levels of inclusion of four grain legumes in broiler diets in a large-scale experiment. Proc. 1998 Aust. Poult. Sci. Symp. 10: 144-147.

Project Title Rapid and specific detection of avian bacterial pathogens RIRDC Project No: DAQ-200A Researcher: Dr Pat Blackall and Ms

Jeanette Miflin Organisation: Dept of Primary Industries

(Qld) Animal Research Institute

Locked Mail Bag No 4 MOOROOKA Q 4105

Contacts: Phone: (07) 3362 9520 Fax: (07) 3362 9429 E-mail: [email protected] Objectives • To develop a rapid and accurate molecular method

for the detection of Pasteurella multocida, the causative agent of fowl cholera.

• To validate, using natural outbreaks of infectious coryza, a polymerase chain reaction method for detection of Haemophilus paragallinarum.

Background Respiratory diseases are a major cause of economic losses to the Australian chicken meat and egg industries. Two bacterial diseases that are important contributors to the respiratory disease complex are fowl cholera and infectious coryza. Both of these diseases present considerable challenges for diagnostic laboratories. The development of tools to assist in rapid and accurate disease diagnosis will be of considerable benefit to industry, allowing the development of effective, targeted and sustainable prevention and control programs.




Research A polymerase chain (PCR) test for P. multocida was developed using primers based on the 23S ribosomal RNA gene. The test is capable of detecting a wide range of reference strains and field isolates of avian P. multocida. The test does not give false positive reactions with any other organisms commonly found in poultry. Two PCR tests for H. paragallinarum were validated in several pen trials and three field outbreaks. Both tests performed as well as the traditional cultural method on swabs of mucus taken from the sinuses of live chickens suffering from infectious coryza but gave results more rapidly. The most significant finding of the research work was that the PCR tests clearly outperformed the traditional culture method when used on samples obtained from coryza outbreaks in commercial chickens. Outcomes Industry now has rapid, accurate and validated tests available to help in the diagnosis of fowl cholera and infectious coryza. Implications Both the chicken meat and egg industries have a goal of maximising production by minimising the effects of infectious diseases on flock health. This goal requires the ability to rapidly and accurately diagnose disease outbreaks. The diagnostic tests developed and validated in this project will help to meet those needs. Project Title Sabbatical studies – Dr Mazhar Khan: Development of molecular tests for serovar-specific identification and typing of Haemophilus paragallinarum RIRDC Project No: DAQ-226A Researcher: Dr Pat Blackall and

Dr Mazhar Khan Organisation: Dept of Primary Industries

(Qld) Animal Research Institute

Locked Mail Bag 4 MOOROOKA QLD 4105

Contacts: Phone: (07) 3362 9498 Fax: (07) 3362 9429 E-mail: [email protected] Objectives • To develop a DNA-based alternative to the

traditional serotyping of H. paragallinarum. • To develop a rapid PCR-based fingerprinting

technique suitable for use as a typing tool for H. paragallinarum.

Background Infectious coryza, caused by the bacteria Haemophilus paragallinarum, is an acute respiratory disease of chickens. In modern intensive poultry industries the major economic effect of the disease is an increased culling rate in meat chickens and a drop in egg

production in laying and breeding hens. While major advances have recently been made which have greatly improved our ability to diagnose infectious coryza, several problems exist which restrict the development of effective prevention and control programs for the disease. An alternative to traditional serotyping of H. paragallinarum is needed, as serotyping of this pathogen is demanding and available at very few laboratories. Improved and less demanding techniques for fine typing of H. paragallinarum strains would also greatly assist in investigating the epidemiology of infectious coryza outbreaks. Research Two different types of rapid, DNA-based typing methods were evaluated for their ability to recognise subtypes within the species Haemophilus paragallinarum. The two techniques both utilised PCR technology. One of the techniques was based on the use of random DNA sequences termed RAPD. The other technique was based on DNA sequences that were originally discovered in bacteria that belong to the family Enterobacteriaceae. This second technique is known as ERIC-PCR. The two techniques were examined from two viewpoints: (i) ability to produce patterns that were serovar

specific ie. an ability to replace conventional serotyping;

(ii) ability to subtype below species and serovar level allowing fingerprinting that is useful for disease outbreak investigations.

Outcomes Despite extensive efforts, the RAPD technique could not be established to a stage where reproducible patterns could be obtained. The technique was thus abandoned. ERIC-PCR did not give serovar-specific patterns and thus cannot replace conventional serotyping. However, this technique was shown to have potential as a typing tool for disease outbreak investigations. ERIC-PCR was shown to have the capacity to clearly distinguish unrelated isolates. The technique was also shown to be as good as some alternative molecular techniques for typing reference strains of H. paragallinarum and field isolates from outbreaks of infectious coryza in China. The very limited genetic diversity of Australian isolates of H. paragallinarum, first identified in previous RIRDC projects, was confirmed with ERIC-PCR. ERIC-PCR was unable to separate the 16 Australian isolates of H. paragallinarum. Implications This project has validated ERIC-PCR as a molecular typing tool for investigating outbreaks of infectious coryza. The technique, however, is of limited application in Australia because of the unique nature of the limited genetic diversity of Australian isolates of H. paragallinarum. This study has shown that, for Australian outbreaks of infectious coryza, the definitive typing technique remains the demanding and time-consuming technique of restriction endonuclease analysis.




Project Title Development of standard MDV challenge viruses in cell culture RIRDC Project No: DAV-115A Researcher: Dr Robin Condron Organisation: Dept of Natural Resources and


475-485 Mickleham Road ATTWOOD VIC 3049 Contacts: Phone: (03) 9217 4200 Fax: (03) 9217 4299 E-mail: [email protected] Objective • To characterise two field MDV strains in

lymphocyte or tissue culture preparations in terms of their pathogenicity.

Background Evaluation of the protection provided by potential vaccine candidates for Marek’s disease requires a reproducible and standard virulent challenge. In recent Australian challenge studies, virus produced in lymphocyte preparations have been used. Virus in these preparations has presented difficulties because the virus is not readily titrated in vitro before the commencement of challenge experiments. A standard challenge system could be developed by seed-lotting low passage tissue culture adapted virus. This would provide better standardisation by titration of the preparation in cell culture prior to inoculation and would provide for relatively easy and rapid preparation of virus without requiring chicken experiments and lymphocyte harvest. Research Groups of specific pathogen free (SPF) chickens were challenged with virulent Marek’s disease virus derived from Woodlands No.1 strain or from isolate MPF 57 prepared in cell culture or in a lymphocyte preparation. Three concentrations of each challenge virus in cell culture were compared with the lymphocyte preparation. Birds were necropsied at either four weeks or ten weeks post inoculation and examined for gross or histological lesions and viraemia. Ratios of bursa weight to bodyweight were calculated. Outcomes All preparations of challenge virus were virulent and produced greater than 80% death or gross lesions. Cell culture grown virus provided better standardisation of inoculum dose and there was no appreciable loss of virulence in comparison with lymphocyte preparations. Both lymphocyte and cell culture grown MPF 57 virus and Woodlands No.1 strain were virulent and produced acute Marek’s disease. Implications A dose of 102 pfu/bird was subsequently proposed for both the Woodlands No.1 or MPF 57 challenge viruses in

cell culture for future challenge studies. This will allow for improved standardisation of future evaluations of the efficacy of Marek’s disease vaccines available or under development in Australia. Project Title A comparison of total and digestible amino acids in diets for broilers and layers RIRDC Project No: UQ-52A Researcher: A/Prof David J Farrell and

Mr Paul Mannion Organisation: University of Queensland Dept of Agriculture ST LUCIA QLD 4072 Contacts: Phone: (07) 3365 2051 Fax: (07) 3365 1177 E-mail: [email protected] Objectives • To measure the chemical composition of two feed

grains and nine protein concentrates and determine the digestibility of their amino acids using caecetomised adult cockerels and excreta collection, following force-feeding.

• To compare the production of hens and growth rate of broiler chickens on diets formulated on a total and digestible amino acid basis and to identify any economic benefits.

• To determine the total and digestible amino acid requirements of starter broilers to 24 days of age.

Background There is considerable debate as to the merits of formulating poultry diets on a total or digestible amino acid basis. There is also debate regarding the method used to measure in vivo the amino acid digestibility of feed ingredients as there are several options. It is known that some feedstuffs give a low digestibility coefficient for some essential amino acids eg. cottonseed meal and heat-damaged proteins. Most feed ingredients have comparatively high amino acid digestibility values (>80%), thus when formulating diets on a total amino acid basis this difference of <20% is normally taken into account by the nutritionist. There have been few experiments that have been designed to test the hypothesis that formulating diets on the basis of digestible rather than on a total amino acid basis gives an economic advantage when using conventional feeding stuffs. This was the basis of the experiments undertaken within this project. Threonine is an important essential amino acid in poultry diets. With changes in genotypes that grow faster and with improved food efficiency there is a need to re-evaluate broiler requirements for threonine. This was done with broilers grown to 24 days of age.





Research Small quantities of eleven feed ingredients were transported to France to be assayed for chemical composition and total digestible amino acids using standard procedures. A modified Sibbald method was used with caecetomised adult cockerels. The force-fed birds gave a true digestible amino acid profile for each ingredient. Apparent metabolisable energy of diets was determined using laying hens and total collection over four days (classical method). There were two practical type layer experiments in which diets were formulated to 100% and 85% of total and digestible amino acid basis (experiment 1) or to 97% and 90% basis (experiment 2). In a large-scale broiler trial diets were also formulated to a total or digestible amino acid basis ranging from 100% to 91% of requirements in increments of 3%. In determining total and digestible threonine requirements, a summit diet and a basal diet were blended to give levels that ranged from well below to well above requirements. These were offered separately to male or female broiler chicks from 3 to 24 days of age. Outcomes Analyses of the feed ingredients agreed with those reported in the literature. With few exceptions amino acid digestibility coefficients were high. However, cottonseed meal and one meat and bone meal gave generally lower values than the other ingredients. The first layer experiment gave no differences in any production parameter between diets formulated on a total or digestible amino acid basis at 100% of requirements. The broiler growth trial showed no differences in growth rate and feed conversion ratio (FCR) at 42 days between diets. Since there was no sex × diet interaction, groups were combined for each diet. In all cases there was tendency for diets formulated on a total rather than a digestible amino acid basis to give better performance. Total and digestible threonine requirements were higher than those previously reported. The actual value varied depending on whether growth rate or FCR was used as the parameter for responses. Implications The results of the experiments in which digestible and total amino acid formulations were used suggest that nutrient specifications for layer and broiler diets may be too high. With conventional ingredients, there does not seem to be any clear advantage in using digestible rather than total amino acid values. If anything, total gave a non-significant advantage over digestible in the broiler experiment, although the diet formulated to 90% of digestible amino acid specifications gave the lowest cost ($/kg eggs) in experiment two. Caution must be exercised when interpreting some of these results because of the method of determining amino acid digestibility. The use of excreta for amino acid

analysis even in caecetomised adult birds raises some doubts. Finally, the recent genotypes of broiler that grow more rapidly, with lower FCRs, than previously would be expected to have increased amino acid requirements. This was shown to be true for threonine but this increase should be evaluated on an economic basis by feed formulators. Publications Mannion, P.F., Perez-Maldonado, R.A. and Farrell, D.J.

(1998) Responses of meat chickens to diets differing in total and digestible threonine content. Proc. 1998 Aust. Poult. Sci. Symp. 10: 136-139.

Project Title Novel strategies for improved mucosal immunity in chickens - application to control of salmonellosis; and Junior Research Fellowship - Ms Wendy Muir RIRDC Project No: US-62CM and

US-64CM /US-44E Researchers: Prof Alan J Husband,

Ms Wendy Muir, A/Prof Wayne L Bryden

Organisation: University of Sydney Dept of Veterinary Anatomy and Pathology

SYDNEY NSW 2006 Contacts: Phone: (02) 9351 7130 Fax: (02) 9351 7348 E-mail: [email protected] Objective • To identify vaccination strategies which induce IgA

predominant intestinal mucosal immune responses in chickens to non-replicating antigens. Successful vaccination techniques were evaluated for their ability to reduce Salmonella Typhimurium infection in immunised chickens.

Background The intestinal mucosa is a common site of entry of major poultry pathogens such as S. Typhimurium and Campylobacter jejuni. Immune defence at the intestinal mucosa is characterised by preferential production of IgA antibody. Due to a limited understanding of the avian intestinal immune system, inappropriate, empirical immunisation procedures have in the past been used to stimulate an intestinal immune response in an attempt to protect chickens from such pathogens. This project was designed to more fully elucidate the function of the avian intestinal immune system and to identify immunisation regimens that specifically stimulate IgA production at the intestinal surface, providing improved protection from intestinal pathogens.




Research Adoptive transfer of intestinal immune cells enabled identification of areas of localisation of cells capable of producing antibody, that is B cells. The quantity of IgA produced following immunisation with either injected and/or orally delivered non-replicating antigen to these sites was compared. The most efficacious immunisation procedure, in terms of IgA production, was then used for vaccination with whole killed S. Typhimurium. Following a challenge from the same isolate, the degree of S. Typhimurium infection was determined. Outcomes The ileal lymphoid aggregate and caecal tonsils were found to contain significant numbers of B cells. A primary intraperitoneal (IP) immunisation followed with an oral booster induced significant intestinal IgA antibody titres, specifically directed against S. Typhimurium. This immune response provided partial protection from a challenge of homologous S. Typhimurium.

Implications IP immunisation induced notable production of IgA antibody at the intestinal site against the non-replicating antigen of interest. Hence, this immunisation procedure may be used to improve and manipulate intestinal immune responses in chickens. However, the protection that this provides may vary, depending on the target pathogen. Novel strategies for oral delivery of non-replicating antigens require optimisation for use in the chicken. Publications Muir, W.I., Husband, A.J., Gipps, E.M. and Bradley

M.P. (1994) Induction of specific IgA responses after oral vaccination with biodegradable microspheres containing a recombinant protein. Immunol. Lett. 42: 203.

Muir, W.I., Bryden, W.L. and Husband, A.J. (1995) Intraperitoneal immunisation promotes local intestinal immunity in chickens. Avian Path. 24: 679.

Muir, W.I., Bryden, W.L. and Husband, A.J. (1998). Comparisons of S. Typhimurium challenge models in chickens. Avian Diseases (In press).

Muir, W.I., (1996) PhD Thesis, University of Sydney: Novel strategies for improved mucosal immunity in chickens.



3.1 & 3.2 CHICKEN MEAT & EGG INDUSTRY

JOINT RESEARCH IN PROGRESS Project Title Diagnostic tools for detection of vvIBDV strains and characterisation of Australian variants RIRDC Project No: CSA-2A Start Date: 1 July, 1997 Finish Date: 30 June, 2000 Researcher: Dr Jagoda Ignjatovic Organisation: CSIRO Division of Animal

Health Private Bag No 24

GEELONG VIC 3052 Contacts: Phone: (03) 5227 5000

Fax: (03) 5227 5555 E-mail: [email protected] Objectives • To develop diagnostic tools to detect exposure of

local poultry to very virulent (vv) IBDV strains, where the birds are already immune to endemic strains.

• To determine the nucleotide sequence of vvIBDV strains from neighbouring countries to assess the genetic diversity of these viruses and assure the detection of all strains.

• To further characterise Australian variant IBDV strains which differ from the overseas variants, in order to obtain a better understanding of the signficance of changes detected in local strains.

Current Progress Antigenic characterisation of infectious bursal disease virus (IBDV) stains isolated from 16 poultry farms in five States has indicated that antigenic IBDV variants are currently restricted in their distribution to Victoria and South Australia. All variants isolated in Victoria were similar but differed from those isolated in South Australia. In the other three States only classical IBDV strains similar to vaccine strains have been isolated. A chicken recombinant antibody library was generated from chicks immunised with 002/73 vaccine strain. From this library two diverse antibodies were selected. One was specific, reacting in ELISA with only three vaccine strains, 002/73, Bursavac live and Bursavac killed, and one field strain from NSW, 06/95. This antibody was produced as a soluble antibody and showed neutralising activity in ova as well as in tissue culture. Following consultation with the poultry industry and other relevant bodies, an approval has been granted to CSIRO for the importation of very virulent IBDV (vvIBDV) strains into the high containment area of the Australian Animal Health Laboratory. Reference vvIBDV strain CS88, virulent strain 52/70 and low virulence strain 1/68 have been imported from the UK. In addition a number of IBDV isolates suspected to be vvIBDV strains have been imported from Indonesia. These strains will be used in

the future development of diagnostic tools for detection of vvIBDV. Project Title Field evaluation of mass vaccination techniques using V4 and heat resistant V4 (HRV4) Newcastle disease virus vaccine strains on caged layers RIRDC Project No: DAN-130A Start Date: 1 July, 1995 Finish Date: 30 September, 1998 Researcher: Dr George Arzey Organisation: NSW Agriculture

Elizabeth Macarthur Agricultural Institute PMB 8


Fax: (02) 4640 6429 Objective • To demonstrate the efficacy of the Australian

Newcastle disease vaccines (V4 & HRV4) applied at different ages by the oral route (drinking water), to induce flock mean HI titres of 3log 2 or higher in commercial caged layers.

Current Progress The project established that the imported strains of caged layers responded poorly to drinking water vaccination with V4 regardless of the dose rate. Drinking water vaccination using a dose rate of 107 EID50/bird, followed one week later by intramuscular vaccination with live V4 at a dose rate of 108 EID50/bird, was identified in earlier experiments as the approach most likely to result in antibody levels adequate to protect laying hens against Newcastle disease (HI titre of 3log2 or higher). Results from trials in which this vaccination regimen was applied ranged from a flock mean titre HI of 3log2 in one flock to 3.7log2, 3.8log2 and 4.1log2 in the other flocks. In all vaccinated flocks mean HI titres reached the minimum target HI level. An additional flock, naturally exposed to V4, was vaccinated by the intramuscular route with live V4 using 108 EID50/bird. This produced a log increase in mean HI titres. This demonstrates that layer flocks, regardless of their Newcastle disease virus status, are likely to respond adequately to the regimen used in these experiments. It is also useful to consider this information in the context of the debate on the reliance on a rise in HI titres in overseas flocks (vaccinated against ND) if exposed to a mild strain of ND.




Project Title Improved control strategies for Marek's disease RIRDC Project No: DAQ-28CM and DAQ-24E Start Date: 1 July, 1993 Finish Date: 30 June, 1996 Researcher: Dr Peter Young Organisation: Dept of Primary Industries

(Qld) Animal Research Institute Locked Mail Bag 4 MOOROOKA QLD 4105


E-mail: [email protected] Objectives • To develop type specific assays for Marek's disease

virus (MDV) and antibody. • To use these assays to:

(a) investigate the epidemiology of Marek's disease in commercial operations; and

(b) optimise the use of currently available vaccines. • To use this information to develop and evaluate

management programs which will reduce disease and provide maximum immunity with currently available vaccines.

Current Progress Type-specific polymerase chain reaction (PCR) tests have been developed for Type 1 (virulent) and Type 3 (HVT vaccine) strains of Marek's disease virus. These procedures have been demonstrated to work on separated blood lymphocytes, tumour and feather follicle sample material. A procedure has been developed where a drop of whole blood, collected onto a filter disk, can be used for the PCR. Research undertaken has also shown that the number of 132 bp repeat regions in the genome of Marek’s disease virus is not correlated with virulence as has been proposed by American workers. For detection of post-vaccination antibody, an ELISA test has been developed which should be useful in determining if flocks have been adequately vaccinated. Project Title Marek's disease challenge trials RIRDC Project No: DAQ-31CM and DAQ-29E Start Date: 1 March, 1995 Finish Date: 30 June, 1995 Researcher: Dr Peter Young Organisation: Dept of Primary Industries



E-mail: [email protected]

Objective • To establish the efficacy of currently available

vaccines against local Marek's disease isolates in the presence of maternal antibody.

Current Progress A series of vaccination trials were carried out in flexible film isolators, using SPF (specific pathogen free) chicks as well as commercial layer and broiler birds. Commercial birds had maternal antibody to Marek's disease as a result of parental vaccination. The vaccines used were Australian commercial Type 2 and Type 3 (HVT). Either alone or in combination, the efficacy of these vaccines was considerably diminished by the presence of maternal antibody. Vaccine registration trials are usually carried out in SPF birds where protective indices of 80% are common. In birds with maternal antibody the same vaccine(s) provided inadequate protection against challenge with virulent Type 1 virus, giving protective indices of less than 60%. In some trials, no protective benefit could be demonstrated for the Type 2 vaccine used alone. Project Title Marek's disease challenge trial 4 RIRDC Project No: DAQ-212A Start Date: 1 July, 1996 Finish Date: 30 November, 1996 Researcher: Dr Peter Young Organisation: Dept of Primary Industries



E-mail: [email protected] Objective • To establish the efficacy of currently available local

and imported vaccines, alone and in combination, against challenge by virulent Type 1 Marek's disease virus in the presence of Type 1 and Type 3 maternal antibody.

Current Progress Vaccination trials in flexible film isolators were carried out to compare the efficacy of an imported Type 1 (C/R6) vaccine with currently available local vaccines, either alone or in various combinations. Both HVT and C/R6 provided good protection in SPF (specific pathogen free) birds, but protective indices were considerably reduced in commercial layer birds with maternal antibody. The best vaccine combinations were either HVT + C/R6 or HVT + Maravac (an Australian Type 2 vaccine). The trials did not provide good support for the introduction and use of C/R6 to control Marek's disease in Australian flocks.






Project Title Attenuation and characterisation of Eimeria species for use in a living vaccine for avian coccidiosis RIRDC Project No: DAQ-215A Start Date: 1 July, 1996 Finish Date: 30 June, 1999 Researcher: Dr Wayne Jorgensen Organisation: Dept of Primary Industries

(Qld) Animal Research Institute Locked Mail Bag 4

MOOROOKA QLD 4105 Contacts: Phone: (07) 3362 9455

Fax: (07) 3362 9429 Objectives • To isolate and purify field strains of Eimeria tenella

and Eimeria necatrix. • To modify the prepatent period of one strain of each

species by selecting for precocious development. • To characterise these modified strains in terms of

their drug sensitivity, reproductive potential, pathogenicity and protection against homologous and heterologous challenge.

• To identify and apply DNA-based techniques to differentiate E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, E. praecos and E. mitis.

• To collect and purify a bank of field isolates of the avian Eimeria.

Current Progress Seven strains of E. tenella and five of E. necatrix have been isolated, purified and cryopreserved. The prepatent period of two strains of E. tenella and one of E. necatrix has been modified by selecting for precocious development. The prepatent period of a second strain of E. necatrix was not successfully modified, however modification of a backup strain of E. necatrix is underway. One modified strain of E. tenella has been characterised in terms of its drug sensitivity, reproductive potential, pathogenicity and protection against homologous and heterologous challenge. One modified strain of E. tenella and one of E. necatrix are in the process of being characterised. DNA techniques to identify E. acervulina, E. tenella and E. mitis have been developed and have been successfully applied to identify species in field submissions. Development of identification techniques for the remaining four species is underway. A bank containing several purified, cryopreserved field isolates of each of the Australian species of poultry Eimeria has been developed and will continually be added to during the course of the project.

Project Title Raw soybeans selected for low trypsin inhibitor activity for poultry diets RIRDC Project No: DAQ-228A Start Date: 1 July, 1997 Finish Date: 31 December, 1998 Researcher: Dr Rider A Perez-Maldonado Organisation: Dept of Primary Industries

(Qld) Queensland Poultry Research and Development Centre PO Box 327

CLEVELAND QLD 4163 Contacts: Phone: (07) 3824 3081

Fax: (07) 3824 4316 E-mail: [email protected] Objectives • To evaluate new low trypsin-inhibitor soybean

genotypes as a low cost source of high quality protein and energy for the poultry industries.

• To make recommendations to the poultry industries on the nutritional value of these genotypes and hence their commercial value in least-cost diets.

Current Progress Full-fat soybeans (FFSB) are an excellent source of high quality protein (37-42%) and a rich source of energy due to their oil content (18-22%). Unfortunately FFSB contain trypsin-inhibitors (called Kunitz and Bowman-Birk) which decrease pancreatic trypsin and chymotrypsin activities thereby reducing animal performance. Fortunately the trypsin-inhibitors can be denatured by heat but processing is expensive and reduces the cost-competitiveness of FFSB relative to solvent extracted soybean meal in broiler diets. A new genotype of soybean, which appears to lack the Kunitz trypsin-inhibitor allele (Kti), has been developed by CSIRO. In a study with broiler chickens the performance of Kti soybean was superior to that of raw soybean but poorer than that of two commercial heat-processed soybean treatments. Data for pancreas weight suggests that these differences were due to residual trypsin-inhibitor levels in the soybeans. A metabolisable energy (AME) assay, however, showed the AME of the Kti soybean to be comparable with that of a commercial FFSB meal and superior to that of raw soybean. Further studies will investigate the effects of steam-pelleting diets with Kti soybean and will include an assessment with laying hens.




Project Title Marek’s disease vaccine seed attenuation and challenge trial RIRDC Project No: DAV-125A Start Date: 1 July, 1996 Finish Date: 11 October, 1996 Researcher: Dr Robin Condron Organisation: Dept of Natural Resources and


475-485 Mickleham Road ATTWOOD VIC 3049 Contacts: Phone: (03) 9217 4200 Fax: (03) 9217 4299 E-mail: [email protected] Objectives • To assess the degree of attenuation of several

Marek’s disease vaccine seeds by comparison to the virulent parent strain Woodlands No.1.

• To determine the protection provided by these vaccine strains against challenge by the virulent parent strain.

Current Progress Four Marek’s disease type 1 vaccine candidates were tested for their efficacy in specific pathogen free (SPF) chickens. These vaccine candidates had been prepared by Prof Greg Tannock and Mr David DeLaney at the Royal Melbourne Institute of Technology and were derived from various passage levels (63, 68, 73 and 78) of the Woodlands No.1 Marek’s disease virus. All vaccine candidates were found to be protective against challenge with the Woodlands No.1 parent virus. However all exhibited some residual virulence as demonstrated by body weights and/or bursa to bodyweight ratios. Pseudemonas infection was apparent in a proportion of the birds in all groups receiving any of the vaccine candidates. The vaccine candidate that appeared to be the most attenuated (RMIT 78) was selected for further evaluation for its vaccine potential. Project Title Three vaccine trials on Marek’s disease RIRDC Project No: DAV-145A Start Date: 1 June, 1997 Finish Date: 31 March, 1998 Researcher: Dr Robin Condron Organisation: Dept of Natural Resources and


475-485 Mickleham Road ATTWOOD VIC 3049

Contacts: Phone: (03) 9217 4200 Fax: (03) 9217 4299 E-mail: [email protected] Objectives • To determine the safety of the RMIT vaccine

candidate MDV Type 1 vaccine at various doses. • To determine the protective dose 50% (PD50) of the

RMIT candidate MDV Type 1 vaccine. • To determine the efficacy of various vaccination

strategies including existing local and imported vaccines and the RMIT candidate MDV Type 1 vaccine.

Current Progress In a large scale safety trial, the RMIT Type 1 vaccine, even when used at high doses, did not cause gross lesions or tumours and appeared to have little effect on the bursa and thymus of vaccinated chickens. Bursa to bodyweight ratios were found to be moderately lower in vaccinated birds than in unvaccinated birds. A dermatitis syndrome noted in 30-40% of vaccinated birds in an earlier, smaller scale study, was only apparent in 5% of vaccinated birds in this trial. Birds with dermatitis also exhibited bursa and thymic atrophy. The 50% protective dose, or PD50 (the concentration of vaccine virus required to induce protection in 50% of vaccinated chickens) of the RMIT vaccine was determined to be 97.7 PFU. In a trial in specific pathogen free (SPF) chickens in which the RMIT vaccine was compared with other commercial vaccines, the highest rates of protection were obtained with the commercial Rispens vaccine used alone (protective index of 97.7%), or Rispens used in combination with the TMC HVT vaccine (protective index of 95.5%). However, the RMIT vaccine when used in combination with TMC HVT also provided very satisfactory protection (protective index of 92.4%). When the mean bursa to bodyweight ratios of the vaccine groups were compared, the Rispens vaccine again performed the best, resulting in higher ratios. The results of these trials indicate that the RMIT vaccine is relatively safe and efficacious in SPF chickens. However, the vaccine comparisons need to be repeated in commercial chickens. Project Title Improving feed grains quality RIRDC Project No: GRD-1A Start Date: 15 April, 1997 Finish Date: 30 June, 2000 Researcher: Dr John Black Organisation: Grains Research &

Development Corporation c/- John L Black Consulting Locked Bag 21





WARRIMOO NSW 2774 Contacts: Phone: (02) 4753 6231 Mobile: 0419 493 567

Fax: (02) 4753 6295 E-mail: [email protected] Objectives A major research program for improving feed grains quality and marketing has been negotiated in response to identified industry needs. The project receives collaborative funding from the Grains Research and Development Corporation and the various intensive livestock RDC’s. It has five integrated component Projects. • Coordination. • Production, storage and distribution of grain

samples. • Rapid and objective analytical tests for assessing

feed grains quality. • Enhancing grain nutritional value through breeding

and processing. • Modelling feed grain quality. Current Progress Seventeen literature reviews were commissioned to collate knowledge on the chemical and physical characteristics of grains that were thought to determine their nutritional value for poultry, pigs and ruminants. The reviews also recommended the best analytical methods for determining these important characteristics and for the in vitro and in vivo measurement of the energy and amino acids available for each livestock species. Approximately 700 cereal grain samples from plant breeders and other sources have been collected and scanned by near-infra-red spectroscopy (NIR). Sixty grains, covering the full range in NIR spectra, have been selected for growing-out twice yearly to provide sufficient material for physical and chemical characterisation and for subsequent in vitro and in vivo trials. Other grains that are found to vary widely in nutritional value will be collected on an opportunistic basis. A selection of grains covering the widest possible range in characteristics thought to determine nutritional value will be fed to poultry, pigs and cattle. Experiments have commenced measuring the AME and available amino acid content of 20 grains in broiler and adult birds as well as in pigs and sheep. A feasibility study on the need for development of analytical kits to identify chemical residues, mycotoxins and weed seed toxins in grains has been commissioned. Project Title Development of Marek's disease Type 1 vaccine RIRDC Project No: RMIT-4CM and RMIT-12E Start Date: 1 January, 1994 Finish Date: 30 June, 1997 Researcher: Prof Greg Tannock Organisation: Royal Melbourne Institute of

Technology

GPO Box 2476V MELBOURNE VIC 3001


E-mail: [email protected] Objectives • To characterise and attenuate, by passage under

appropriate GMP conditions, a virulent Australian MD Type 1 virus.

• To evaluate passage levels beyond P20 for attenuation and efficacy against virulent Australian MD Type 1 challenge.

• To undertake tasks and testing required of a suitable candidate virus to establish a seed lot system for vaccine production.

• To undertake studies to optimise growth of candidate vaccine virus.

• To undertake dose response and other studies required to determine usage of virus as a vaccine.

Current Progress The project has proceeded to a stage at which large scale field trials comparing an RMIT vaccine candidate with vaccines prepared from the imported Rispens vaccine seed strain can be carried out (see report on project RMI-6A). Work undertaken during the last twelve months has been directed towards determining a passage level of the Woodlands No.1 Marek's disease virus strain in chicken embryo fibroblast cultures at which an optimal balance between avirulence and immunogenicity is achieved for the RMIT vaccine. These studies have been carried out in specific pathogen free (SPF) birds at the Victorian Institute of Animal Science. In previous years, because of problems in standardising the challenge used in bird experiments, two highly virulent Australian field viruses were grown to a limited number of passages in cell culture and maintained as seed lots for use in the development of an Australian vaccine. These viruses are available for general use and will be described in the journal "Avian Pathology" in October 1998. Project Title The development of effective immunisation strategies against Marek's disease RIRDC Project No: RMI-6A Start Date: 1 July, 1997 Finish Date: 30 June, 2000 Researcher: Prof Greg Tannock Organisation: Royal Melbourne Institute of

Technology GPO Box 2476V

MELBOURNE VIC 3001 Contacts: Phone: (03) 9925 3088







Objectives • To characterise and attenuate, by passage under

appropriate GMP conditions, a virulent Australian MD Type 1 virus.

• To evaluate passage levels beyond P20 for attenuation and efficacy against virulent Australian MD Type 1 challenge.

• To undertake tasks and testing required of a suitable candidate virus to establish a seed lot system for vaccine production.

• To undertake studies to optimise growth of candidate vaccine virus.

• To undertake dose response and other studies required to determine usage of virus as a vaccine.

Current Progress The RMIT serotype 1 vaccine developed under project RMIT-4CM/RMIT-12E was compared with the Rispens vaccine in a trial in specific pathogen free (SPF) birds in late 1997. Results indicate that, when used alone, the RMIT vaccine was slightly less immunogenic than the Rispens vaccine but these differences were not apparent when either of these vaccines were used in combination with HVT vaccine. Studies on the RMIT vaccine indicate that it has similar growth characteristics in cell culture to the Rispens vaccine. In planned future work, this RMIT vaccine will be evaluated in commercial birds in comparison with the Rispens strain. Planning is at present underway to commence a series of studies to enhance immune responses to HVT vaccine. This vaccine has been used in Australia and other countries for over 25 years but appears to offer comparatively little protection against recent very virulent field strains of Marek's disease virus. Preliminary results have indicated that, when HVT was administered to chickens at the same time as a living attenuated Salmonella vaccine, enhanced protection against challenge by very virulent Marek's viruses was obtained, suggesting that the Salmonella vaccine acted as an adjuvant. The role of this Salmonella vaccine will be further evaluated in comparison with other non-bacterial adjuvants, such as gamma inulin. Underlying immunological mechanisms for any enhancement of HVT immunity are being studied. Preliminary results have shown that it is possible to adapt Serotype 1 and HVT vaccine viruses to grow in the Vero continuous cell line. Much higher yields are produced by HVT. Vero-grown preparations of HVT will be compared with preparations of HVT grown in chicken embryo fibroblasts in the SPF challenge system used for the evaluation of Serotype 1 vaccines. Project Title Characterisation of very virulent Australian isolates of Marek's disease virus RIRDC Project No: RMI-8A Start Date: 1 October, 1997 Finish Date: 30 September, 2000

Researcher: Prof Greg Tannock Organisation: Royal Melbourne Institute of

Technology GPO Box 2476V MELBOURNE VIC 3001


E-mail: [email protected] Objectives • To collect and maintain Marek's disease viruses

(MDVs) isolated from flocks in different parts of Australia and to characterise them by molecular and biological tests.

• To determine optimum methods for standardising Australian Marek's disease vaccines.

• To prepare and maintain reference preparations of viruses for use in vaccine assays.

• To provide a vaccine assay facility for use by Australian vaccine manufacturers as and when required and in harmony with requirements set by the National Registration Authority (NRA).

Current Progress Isolates have been obtained from Marek's disease problem flocks in different parts of Australia. Standard protocols for virus isolation in chicken embryo kidney culture that have been developed by the laboratory have been followed but have been modified to include a preliminary identification step using the more sensitive polymerase chain reaction. All viruses isolated have been retained for later characterisation in chicken challenge tests. Assay procedures have been established to estimate infectious titres of Marek's disease vaccines used in Australia. At a meeting held with representatives of vaccine manufacturers and the industry on 17/3/98, agreement was reached on a number of issues relating to the role of the Virology Laboratory at RMIT as a reference centre for Marek's disease vaccine assays. Contact has been made with the National Registration Authority and accreditation of the laboratory will be sought later in 1998. Project Title Development of improved serological diagnosis of Mycoplasma synoviae RIRDC Project No: UM-33A Start Date: 1 July, 1996 Finish Date: 31 December, 1999 Researcher: Dr Glenn Browning Organisation: University of Melbourne

Veterinary Preclinical Centre PARKVILLE VIC 3052 Contacts: Phone: (03) 9344 7342






Objectives • To identify, characterise, clone and express the genes

for M. synoviae specific antigens. • To develop and assess a highly specific serological

test for M. synoviae infections, based on cloned, expressed antigens.

• To improve diagnosis of M. synoviae infection in the field and also enable the accurate assessment of effective vaccination.

• To improve the control of mycoplasmosis in chickens.

• To assess the potential for developing a serologically marked strain of the temperature sensitive MS-H vaccine strain of M. synoviae.

Current Progress The gene encoding two immunodominant species-specific proteins of Mycoplasma synoviae has been identified and characterised. Two regions of this gene have been expressed in E. coli in an expression system which enables easy isolation of the proteins produced. The proteins produced by E. coli are currently being assessed for their suitability for serological diagnosis of M. synoviae infections, both in terms of sensitivity and specificity. Thus the first objective of the project has been met, and current work is addressing the second objective of the project. Project Title Intestinal spirochaete infections in chickens in eastern Australia RIRDC Project No: UMU-21A Start Date: 1 July, 1997 Finish Date: 30 June, 1998 Researcher: A/Prof David Hampson Organisation: Murdoch University

Division of Veterinary and Biomedical Sciences

MURDOCH WA 6150 Contacts: Phone: (08) 9360 2287

Fax: (08) 9310 4144 E-mail: [email protected] Objective • To determine the prevalence and disease associations

of intestinal spirochaetes in Australian layer and broiler breeder flocks.

Current Progress Faecal samples (n=896) from chickens on 36 farms in Queensland, NSW, Victoria, Tasmania and SA have been collected and cultured under anaerobic conditions on selective media suitable for the isolation of intestinal spirochaetes. Overall, spirochaetes were isolated from birds in 15 (42%) of these flocks. Consistent with overseas findings, no birds from nine broiler flocks were colonised. Additional samples are being sought from broiler units, but at this stage it appears that intestinal spirochaetes are unlikely to be a major problem in

broilers in Australia. In contrast, and again consistent with findings overseas and in WA, birds in six of 13 layer (46%) and six of 14 broiler breeder (43%) flocks were infected. Amongst 21 flocks from Queensland, only 6% of samples from farms without intestinal disease contained spirochaetes, whilst 65% of samples from flocks with diarrhoea and reduced production were positive. Representative isolates identified so far have included the chicken pathogens Serpulina intermedia and Serpulina pilosicoli. Additional flocks are being sampled and the identity of individual isolates from diseases and healthy flocks are being determined. Project Title Evaluation of the VICAM Screen/Verify system for detection of Salmonella Enteritidis in poultry flocks RIRDC Project No: UNS-9A Start Date: 1 March, 1996 Finish Date: 30 June, 1997 Researcher: Dr Julian Cox Organisation: University of New South

Wales Dept of Food Science and Technology


Fax: (02) 9385 5931 E-mail: [email protected] Objective • To evaluate the sensitivity and specificity of the SE

Screen/Verify system for detection of Salmonella Enteritidis in poultry flocks.

Current Progress The SE Screen/Verify system consists of a two-step procedure, a genus-level immunomagnetic separation (IMS) and a serovar-specific latex agglutination (LA), for the detection of Salmonella Enteritidis. The sensitivity and specificity of the IMS were assessed. A diverse subset (n = 7) of 73 SE strains varied considerably in the percentage bound (30-100%) by the IMS reagent, suggesting that recovery of SE may be problematic. A selection of these strains was then mixed with either Escherichia coli (EC) or Salmonella Typhimurium (ST) to simulate the presence of closely related background flora likely to be encountered in environmental samples. Separation of SE from EC was significant, evidenced by growth on XLD agar before and after IMS. Some EC were observed, suggesting non-specific adsorption. ST was strongly bound by IMS, indicating that, should salmonellae other than SE be present in a sample and outnumber or outgrow SE, discrimination of SE from other salmonellae would be difficult. The latex agglutination reagent, coated with antibodies believed to react with a fimbrial structure of SE, failed to react with ten of the 73 SE strains. Given the lack of separation afforded by IMS from mixed cultures, LA alone was assessed as a means of detecting SE directly from artificially inoculated, enriched environmental samples.





SE was detected from enrichments in mannitol selenite cystine broth, but not from samples enriched in Rappaport Vassiliadis broth. The use of the LA reagent in this manner shows some promise for routine use, but the potential for false-negative reactions is evident. Project Title Total and digestible tryptophan content of feedstuffs for poultry RIRDC Project No: US-51A Start Date: 1 July, 1997 Finish Date: 30 June, 1999 Researcher: A/Prof Wayne Bryden Organisation: University of Sydney



Fax: (02) 4655 0693 E-mail: [email protected] Objective • To develop a database of the total and digestible

tryptophan contents of feedstuffs for poultry which can be used by the feed industry to formulate more efficient diets.

Current Progress Tryptophan may become a limiting amino acid in Australian poultry diets, especially when diets are based on sorghum, lupins and meat and bone meal. Under this project, a database is being developed of the total concentration and ileal digestibility of tryptophan in Australian feedstuffs for poultry. Tryptophan was not included in the ‘digestible amino acid database’ (RIRDC Publication No. 98/9) published earlier this year, because of the inherent problems in determining this amino acid. While most amino acids are routinely analysed by ion-exchange chromatography following acid hydrolysis, tryptophan is destroyed under these conditions. For this reason, analysis of tryptophan has to be carried out separately and involves additional time and cost. Tryptophan concentrations in feeds and ileal digesta were analysed using a procedure recently established at this laboratory. This procedure involves hydrolysis with sodium hydroxide followed by ion exchange chromatography under isocratic conditions. Thus far, total and digestible tryptophan concentrations of 36 samples representing 12 different feedstuffs have been determined.


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RIRDC Completed Projects in 1997-1998 and Research in ...€¦ · A/Prof David Farrell (07) 3365...

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