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Gene research for pathogen-free chickens

CHICKEN-IMAGE is a three-year project that aims to explore the basis for the genetic immunity of chickens to diseases that are damaging the European poultry production industry. The project is isolating large clusters of genes with functions in immunity and analysing their sequences, to create a new generation of tools for chicken immunology. It will go on to produce a library of relevant gene expression profiles. It also hopes to produce kits for genetic testing that will help breeders to select naturally immune birds so that they can optimise production and profit.

Chickens provide the world with its most important source of animal protein. As the scale of production of the poultry industry has increased, the various pathogens that make chickens sick have become more aggressive. This poses a threat to long-term food supply in Europe and also to the commercial viability of an industry that operates within very limited profit margins.

The Commission responded in 2000 by starting a three-year programme to explore the genetic basis for immunity in chickens and to develop a database of expression profiles for a large set of chicken genes. The ultimate aim is to produce a range of commercially valuable products to provide the technology for breeding resistant chickens. Partners in the programme include research institutes in France, Belgium, Germany, Italy and the UK, as well as a firm of poultry breeders. The project is known as Chicken-Image (Improvement of CHICKEN Immunity and resistance to disease based on Analysis of Gene Expression).

Finding immune gene sites

It might not be thought that chickens have much in common with either mice or people, but at the most basic level of the genome, it seems that there is much shared material. The human and murine genome projects have identified clusters of immunologically important genes that control resistance to a pathogen or affect the course of its development. It is now possible to locate analogous regions in the chicken genome where these genes also occur. The project is mapping novel genes in five such clusters. These are:

  • The natural killer region
  • The CC chemokine cluster
  • The CC chemokine receptor cluster
  • The Selectin/CD1 region
  • The Mhc region.

The genes are identified through physical cloning of the clusters using chicken bacterial artificial chromosomes (BAC) and sequencing the cluster regions in the clones. In this way, large sets of genes can be rapidly identified. This method offers a systematic approach to extracting immunologically relevant genes from the five gene clusters, expanding our understanding of the chicken immune process.

Chicken gene library

Programme participants are working on a database of partially sequenced chicken DNA (cDNA), enriched by material from the cloning experiments. A procedure manual has been produced to ensure that work complies with the established principles of Good Laboratory Practice (GLP); it contains every step needed to produce of full-length sequences for the library, whether normalised or subtracted. Genes have been cloned from various tissues undergoing different kinds of immune responses in infected birds. This approach to identifying genes whose expression is involved in immune responses is likely to supplement more conventional approaches and make a major contribution to the identification of these genes in chickens. This data is available to all the programme participants, and will eventually be put into a public database for both academic and commercial researchers.

Response to infection

Some 15,000 genes are expressed in chicken tissues involved in the regulation and development of immune responses, both innate and adaptive. Part of the project is devoted to generating microarrays on glass representing these genes and identifying the subsets that have relevance to immune response. This has involved collecting all possible data on chicken genes and ESTs and developing analytical methods for selecting the appropriate genes or gene fragments. Precise protocols for reproducing arrays of the selected genes will be vital for effective exploitation in breeding programmes.

A similar approach will investigate the variation in gene expression during the course of actual infection with three chicken diseases, Eimeria tenella, infectious bursal disease and Marek's disease. A comparison of resistant and susceptible birds should -identify genes whose expressions are involved in determining the outcome of an infection. Finally in the last year of the project, the information obtained thus far will be exploited in outbred commercial breeder chickens, and the analyses extended to vaccine responses.

A safe chicken in every pot

The participants hope to use the gene expression profiles to identify patentable gene expression tests . For these tests, quantitative polymerase chain reaction (PCR) assays will be developed for monitoring large numbers of chickens for the levels of expression of specific candidate genes in an efficient way.

Project co-ordinator Dr Zoorob stresses that Chicken-Image is not aiming to produce transgenic chickens. "We hope that we can improve the immunity of chicken flocks genetically, but through traditional breeding methods," she says. "Otherwise the product may not be acceptable to the consumer."

Progress and results

The analysis of the genes present in the BACs spanning the five clusters has been completed. The results for the chemokines and their receptors are consistent and show the inflammatory CC chemokines and their receptors in chickens. These are reduced in number and probably in function, relative to humans and mice. Four cDNA libraries have been constructed and have been normalised. The sequencing of the non- and normalised libraries to identify EST sequences is ongoing. Sustainable agriculture; Plant systems; Animal Systems 45 A specific database for the management and the data mining of considerable information about each clone (sequence, quality, annotation, BLAST results), and a software package (Fountain) have been developed to extend the full coding sequences by primer walk and to assemble the gene contigs.

The project is now in a position to expand the array rapidly, and use the expanded array for analysis of RNA from the proposed animal experiments.
The project included a component of work to identify molecules with immune function on the basis of their expression profiles, as well as by sequence analysis. The project has established in the chicken: a map of five clusters of immunologically important genes with a collection of identified and sequenced genes with important immunological functions, for example a full set of avian chemokines and their receptors a resource of partially and fully sequenced cDNA clones representing immune gene transcripts present in a range of immune tissues.

A database for the management and data mining of information about each clone and a software package (FOUNTAIN) to extend the coding sequences of ESTs by primer walk and to assemble the gene contigs have been developed. The project has led to the construction of immunologically targeted gene arrays for the chicken, consisting of large numbers of genes selected for their involvement in the host response to infection. We have also provided expression profiles in response to infection with IBDV, Eimeria maxima , MDV and a vaccine strain of MDV.

Project Co-ordinator: Dr Rima Zoorob
E-mail: zoorob@infobiogen.fr
Telephone:+33 1 49583500
Address: CNRS-ERS 1984
19 rue Guy Moquet
F-94801 Villejuif
France
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