Immunity is composed of innate and adaptive compartments and parameters can be measured e.g. as antibody titers and the activity of the complement system in the blood. Natural antibodies (NAbs) and the complement system define innate humoral immunity and constitute a substantial part of the basic capacity of an organism to respond to pathogens [¹]. Acquired antibodies neutralize pathogens upon exposure of an individual and are part of the adaptive humoral immunity. NAbs and complement titers have been associated with survival in laying hens [²]. Identification of genes regulating these immunological parameters is of interest, as this information could be used to select for animals with superior immune response.

Feather pecking (FP) is a serious behavioral disorder of laying hens. FP is a multi-factorial problem caused by both genetic and environmental factors. Line differences in FP have been documented [³,⁴]. Feather pecking is heritable [⁵,⁶] and chromosomal regions involved in feather pecking have been identified [⁷,⁸]. In relation to FP and cannibalism social interactions are important and it was shown that the associative effect due to the genotypes of group mates can contribute substantially to the total heritable variation [⁹,¹⁰]. Because measurement of FP is labor intensive, instead feather condition scores was used to assess plumage condition because of the relation to feather pecking activity [¹¹].

A population of 583 hens (for immune parameters) and 660 hens (for feather score) was genotyped for 1022 SNP and used for the association study. The animals came from 4 Rhode Island Red and 5 White Leghorn pure lines. On average 65 hens per line were tested for immunological parameters and 73 hens per line for feather condition score. Phenotypes for immune parameters were described in detail by Star et al. [²]. Feather condition score was previously described by Uitdehaag et al. [⁴].

The approach based on the analysis of multiple lines and tests of SNP-by-line interaction in a population of laying hens measured for several immune traits and feather condition score is described in detail by Biscarini et al. [¹²,¹³]. Among lines, linkage disequilibrium is conserved at shorter distances than in individual lines; therefore, SNP significantly associated with traits across lines are expected to be closer to functional mutations. In the analysis, the SNP that had a significant across-line effect but did not show significant SNP-by-line interaction were identified to test whether the association was consistent in the individual lines [¹⁴].

In this study, we presented an original approach to detect SNP associated with immune and behavioral traits. The method is based on the simultaneous analysis of multiple lines using a multiple-step procedure. In the across-line analysis, the SNP detected were expected to be close the QTL for a trait because of the reduced extent of LD conserved across lines. Building on the work by Saccone et al. [¹⁴], we tested for the SNP-by-line interaction to ensure consistency of the association across lines. A possible source of false positive associations because of population stratification was avoided by including a line effect in the model. Consequently, SNP explaining part of the between-line variation could not be detected in this approach.

The across line analysis was successful in detecting significant associations of SNP for innate immune as well as behavioral traits. The study shows that genetic variants of IL17, IL12, IL10 and IRF1 (located in the proximity of IL4) and the major histo-compatibility complex encoded genes (BLB and TAPBP) affect NAb and/or acquired antibody levels. In addition complement activity of classical or alternative pathway was affected.

The analysis for behavioral traits emphasized the role of the serotonergic system in pecking behavior, the HTR2C gene showed significant association in the direct as well as the associative model. The fact that more significant associations were found in the associative model (n=57), as opposed to the direct model (n=11), is in line with the observation that heritability estimates are higher for performing FP than receiving FP [⁵,⁶]. Associative effects contribute substantially to the total heritable variation, and have been shown to be larger in magnitude than direct genetic effects [⁹,¹⁰].

The relations between behavior and immunity as demonstrated in our analyses by the significant associations with immune regulatory genes, is in line with previous studies by our group [¹⁵]. Low levels of innate immunity, either cellular or humoral, might be related to disease susceptibility, whereas high levels might be related to disease resistance [¹⁶]. This reinforces the notion that Nabs might be important for the maintenance of homeostasis and disease resistance [¹⁷].

The study confirmed previously identified QTL and identified new associations of genes regulating immune function. The results for feather condition score supports existing evidence of involvement of the serotonergic system in feather pecking in laying hens. Immune regulatory genes were found to be associated to feather condition score, revealing relationships between the immune system and behavior.

FP, feather pecking; NAb, natural antibody; SNP, single nucleotide polymorphism.

The authors declare that they have no competing interests.

FB and HB performed the association studies, HP and JvdP were responsible for the immune traits, BR was responsible for the behavioral traits, AJ and JvdP designed and performed SNP genotyping. All authors contributed to the design of the study and revision of the paper.

This work was conducted as part of the SABRETRAIN Project, funded by the Marie Curie Host Fellowships for Early Stage Research Training, as part of the 6th Framework Program of the European Commission.

This article has been published as part of BMC Proceedings Volume 5 Supplement 4, 2011: Proceedings of the International Symposium on Animal Genomics for Animal Health (AGAH 2010). The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/5?issue=S4.