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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_
Genome
Screen
L .. age
M ap
.
genommcs
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enomi
genomics
rait
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Structural_
Variation
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
-_
.'_
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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