83-114). Oxford: Carfax Publishing Company. Klasing, K. (1994). Avian leukocytic cytokines. Poultry Science, 73, 1035-1043. Klasing, K.C. &Korver, D.R. (1997).
Candidate Genes for Immune Response and Disease Resistance in Chickens Susan J. Lamont iowa State University, Department of Animal Science, Ames, Iowa, USA
Abstract Using existing variation in genes that control immunity in poultry is a diseasecontrol approach that is compatible with permanent enhancement of health. Currently, knowledge of genes for host immunity is a limiting factor in effective genetic selection for enhanced disease resistance. Identification of the specific host genes that are crucial in pathways and mechanisms of immunity is, therefore, key to genetic improvement of poultry health. This paper describes several strategies for candidate gene identification for immunity enhancement. Introduction In the USA, costs due to disease prevention, vaccination and medical treatments in poultry are estimated at over $15 billion per year (USDA, 1997). Diseases also have a significant negative impact on efficiency of production; up to 15% of production potential in poultry is estimated to be lost because of disease (Biggs, 1982). Bacterial diseases present significant food-safety problems in human consumption of contaminated, improperly prepared poultry products (meat and eggs), as well as reducing production efficiency in production populations because of the negative biological impact of carrying a bacterial burden (e.g., review by Klasing and Korver, 1997). There are negative consumer reactions to antibiotic use in food animals and the potential introduction of antibiotic-resistant bacteria into the food chain. vim (1997) proposed that changes in the virulence of pathogens, concentration of poultry in larger production units, and failure of pathogen eradication in most commercial operations require efforts to enhance disease resistance by genetic approaches. The inclusion of the genetic approach in a comprehensive program of disease management and production enhancement has many benefits. The genetic improvements represent a long-term and cost-effective solution. Genetic enhancement of immune response increases vaccine efficacy and disease resistance, thereby reducing drug residues in animal products. With poultry producing 40% of the animal products consumed worldwide, it is important to take all possible approaches to enhance production efficiency by maintaining good health in production populations (Sainsbury, 1997). The use of genetic markers for selection is preferable to direct selection on disease traits. Large-scale pathogen challenge testing is costly and environmentally hazardous. Resistance traits are costly and difficult to accurately measure. It is, therefore, preferable to identify genetic markers associated with disease-resistance traits. Optimal use of genetic markers, however, requires that they be able to be effectively used over a wide range of populations. The current lack of knowledge about the specific genes controlling resistance traits limits the effective application of molecular genetic approaches.
26
Identification of Candidate Genes for Immunity and Disease Resistance: The Case of Salmonella enteritidis As a model for the identification of candidate genes for immunity, we examined the specific example of Salmonella. Salmonella enterica Serovar Enteritidis (also known as • Salmonella enteritidis, SE) is an intracellular bacterium that is an important zoonotic pathogen (Saeed et al., 1999). SE has emerged as a worldwide source of food poisoning in humans. Salmonella are transmitted both vertically and horizontally, thereby causing problems at all levels of poultry breeding and production (Lister, 1988; Cason et al., 1994). Infected hens can shed live bacteria into eggs, contaminating both table eggs and chicks. Horizontal transmission of Salmonella can take place from even a very small number of shedders (Byrd et al., 1998). The costs of controlling and preventing SE in hen houses can be up to 2% of the total cost of egg production. Although vaccines and competitive-exclusion treatments exist for SE, their use does not always provide complete protection in the field. : Utilizing a candidate gene approach to define the genetics of Salmonella control in poultry is very feasible, because of the detailed knowledge available in mammals regarding response to primary Salmonella infection (see Figure 1; Sebastiani et al., 1998; Lalmanach and Lantier, 1999; Gruenheid and Gros, 2000; Shiloh and Nathan, 2000; van Deventer, 2000; Eaves-Pyles et al., 2001). The course of a Salmonella infection consists of four distinct phases, each of which involves different effector cell types and molecules. This understanding of molecular and cellular pathways of response in humans and mice serves as a starting point for investigation of the existence of similar mechanisms in chickens, using a functional comparative genomics strategy. Three candidate genes or regions have been previously identified for association with resistance to Salmonella in chickens. Natural resistance-associated macrophage protein 1 (NRAMP1) gene variation accounts for a small percentage of the genetic control of Salmonella burden in spleens alter intravenous inoculation (Hu et al., 1997). A region on chicken chromosome 5, designated SAL1 (Mariani et al., 2001), in an area with no known candidate genes, represents a large component of genetic control. Cotter et al. (1997) reported MHC linkage of resistance to Salmonella-induced mortality. In none of these studies, however, did the identified genes completely account for the genetic control of the resistance, indicating that additional genes exist that contribute to the biological variation in resistance to Salmonella colonization in chickens. It is also clear from studies on mice that multiple genes are involved in innate resistance to Salmonella infection (Sebastiani et al., 1998). In the authoffs lab, genetic line differences and heritability values of S. enteridis antibody levels have demonstrated genetic control ofhumoral immune response to this pathogen in broilers and, therefore, the feasibility of identifying the genetic basis of this trait (Kaiser et al., 1997, 1998). Genetic potential for greater SE vaccine antibody response was associated with lesser SE colonization in unvaccinated, SE-exposed broiler breeder chicks (Lamont, 1998), suggesting that enhancement of innate antibody response levels, as well as vaccine-induced immunity, is important.
Figure
I. Candidate
Genes
for Salmonella
Response
The effectiveness of using the candidate gene approach to dissect the genetic control of complex traits has been reviewed and discussed (Rothschild and Soller, 1997; Tabor et" al., 2002). Not only is this an effective detection strategy but, because the identified polymorphisms are within the causative genes, the general applicability of the results is much greater than for other types of genetic markers. This broad applicability speeds the technology transfer of research results into utilization phases for enhancement of commercial populations. The candidate gene approach is timely, in that many of the sequences of the molecular candidates have recently been identified in chickens. The basic steps in candidate gene analysis are the following: 1. 2. 3. 4. 5.
Select the candidate gene (physiological or positional) Database analysis of genomic organization of candidate gene in another species Design primers from known sequence Sequence PCR product for gene verification Amplify pooled genomic DNA samples from populations to check for polymorphisms 6. Design test for high-throughput amplification and analysis 7. Analyze associations between traits of interest and genotype of candidate gene 8. Analyze gene interactions
Gene E x p re s s io n _"-....._r_
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L .. age M ap
. genommcs
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rait
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Figure
2. Strategies
for Candidate
Gene
Identification
Within our resource population for Salmonella response, the first step of analysis was determination of whether candidate genes associated with response in other chicken populations also were associated with response in the new population. MHC class I variation was evaluated by direct sequencing and the base-excision sequence scanning (BESS) methods (Liu and Lamont, 1999; 2000), and was found to be associated with Salmonella burden in the spleens of challenged chicks. Sequence variation ofNrampl was analyzed and a PCR-RFLP assay was developed to type a previously unevaluated polymorphism. This new polymorphism was determined to be associated with SE response, both bacterial burden and antibody response to vaccination (Lamont et al., 2002). Thus, both candidate genes, MHC and Nrarnp 1, were confirmed as robust over different populations and with different types of Salmonella response, a validation of the functional genomics approach for candidate gene identification (see Figure 2). Prosaposin (PSAP) was identified as a positional comparative candidate gene, because of its location near a microsatellite linked to a QTL for Salmonella response, and its biological role in controlling production of a precursor of glycoproteins that activate lysosomal hydrolysin. PSAP was found to have genetic polymorphisms associated with SE burden in the spleen of challenged chicks (Lamont et aL, 2002), a validation of the positional comparative genomic approach. Two genes in the biological pathway of apoptosis (Inhibitor of Apoptosis- 1, lAP- 1; and Caspase) were evaluated for genetic variation, which was then demonstrated to be associated with Salmonella burden in the spleen, a validation of the functional comparative genomics approach.
Additional categories of genes are likely candidates for investigation in the complex mechanism of genetic control Salmonella. These include genes encoding proteins involved in reactive nitrogen intermediates (Shiloh and Nathan, 2000), cytokine genes and their receptors (Lalmanach and Lantier, 1999; van Deventer, 2000), endotoxin recognition molecules (Eaves-Pyles et al., 2001), and chemokines and antibacterial peptides (see Figure 2). Fortunately, we have moved quickly from a time in which most avian cytokines were defined only by biological activity or protein characterization (Klasing, 1994; Kaiser, 1996) to the gene knowledge of-today. Microan'ay approaches in chickens are currently limited because of the paucity of cell types available in microarrayed format, although this situation is quickly being remedied. Because of the complexity of response to Salmonella, an ESTlibrary representing activated T cells (Tinmagaru et al., 2000), is likely to represerit only a subset of genes involved in Salmonella resistance. However, integration of multiple strategies of genetic analyses is a strong tool to pinpoint candidate genes. Forexample, genomic scans with microsatellites identified genomic regions containing QTL for Marek's disease resistance (Yonash et al., 1999) and comparative gene mapping studies placed genes important for immune function in these same regions (Suchyta et al.,2001). With the advent of microarrays, genes with differential expression levels between resistant and susceptible lines were found and some corresponded to positional or functional candidates for Marek's response (Liu et al., 2001). Summary Knowledge of resistance pathways, derived from any species, and specific information on gene sequence from avian species, enables application of the candidate gene approach to immunity enhancement. Additionally, positional information derived from genomic scans can lead to identification of candidate genes. Likely candidates for genetic control of resistance to many bacterial diseases include the cytokine genes, the genes of the major histocompatibility complex, and genes involved in apoptosis, endotoxin recognition and bactedocidal activity. Careful evaluation of biological response mechanisms in the species of interest or in other species, coupled with detailed evaluation of sequence variation in appropriately designed and measured resource populations can yield highly successful outcomes in candidate gene searches. An integration of candidate gene analysis, gene expression profiling, and genomic screening approaches presents the most powerful and comprehensive approach to enhance poultry immunity and health by genetic means. Acknowledgments
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The Lamont research program is supported by State of Iowa, Animal Health and Multistate Research funds, poultry breeding companies, and grants from BARD, a Cargill Research Excellence Fellowship and the Midwest Poultry Research Program. Major Salmonella project researchers are: Mike Kaiser, Wei Liu, and Massoud Malek.
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