Transgenerational epigenetic effects on innate immunity in broilers: An underestimated field to be explored?

Transgenerational epigenetics is becoming more and more important for understanding the variation of physiological responses of individuals to the environment and the inheritance of these responses based on all mechanisms other than the actual DNA nucleotide sequence. Transgenerational epigenetics is the phenomenon that the information of the environment of (usually) a female animal is translated into memory-like responses preparing the offspring. As a consequence, individuals of the next generation may show different phenotypic traits depending whether their mothers were kept under different environmental conditions. This may result in either positive or negative effects on the next-generation individuals, which is different from individuals from mothers that have been kept in a different environment. Transgenerational epigenetic effects have been proposed and indicated for specific immune (T cell and antibody) responses (especially in mammals, but also in birds) and innate immunity (nonvertebrates), but surprisingly very little is known of transgenerational effects on innate immunity in chickens. Given the short lifespan of the chicken and therefore the likely dependence of chicken on innate immune mechanisms, more attention should be given to this arm of immunity and mechanisms of inheritance including transgenerational effects that can be initiated in the breeder generation. In addition, it is becoming evident that innate immunity also underlies metabolic disorders in broilers. In the current paper, we will argue that although very little is known of transgenerational effects of innate immunity in poultry, more attention should be given to this type of study. We will illustrate examples of transgenerational epigenetics, and finally propose strategies that should reveal the presence of transgenerational epigenetic effects on innate immunity in chickens and strategies to modulate breeder birds such that these effects positively affect innate immunity of broilers. It is suggested that a mismatch between breeder environment and broiler environment may account for unwanted effects of innate immunity in the broile

Body weight deviations as indicator for resilience in layer chickens

Resilience is the capacity of an animal to be minimally affected by disturbances or to rapidly return to the state pertained before exposure to a disturbance. Less resilient animals are expected to be more susceptible to environmental perturbations, such as diseases, and will consequently show more and/or greater fluctuations in production than more resilient animals. Natural antibodies (NAb) are antibodies recognizing antigens without previous exposure to these, and are hypothesized to be an indication of general disease resistance. The objective of this research was to investigate genetic parameters of resilience indicators based on standardized body weight (BW) deviations and to investigate its relation with immunity (i.e. NAb) and disease resistance. Keyhole limpet hemocyanin-binding NAb were measured in layer chickens, which were selectively bred for high and low keyhole limpet hemocyanin-binding NAb levels during six generations. In addition, BW data of these layers were collected on a four-weekly interval from 4 weeks of age until 32 weeks of age. Standardized deviations of BW from an individual were compared to lines’ average BW (i.e. across individuals), and these were used to calculate resilience indicators: natural logarithm-transformed variance [ln(variance)], skewness, and lag-one autocorrelation of deviations (i.e. all within an individual). Heritabilities of resilience indicators were between 0.09 and 0.11. Genetic correlations between the three resilience indicators were between -0.20 and 0.40 (with high SE), which might suggest that the resilience indicators capture different aspects of resilience. Genetic correlations between resilience indicators and NAb were close to zero, which suggests that the resilience indicators and NAb capture different aspects of immunity. This might indicate that, in this dataset, environmental perturbations are only to a small extent affected by disease incidence, possibly due to a lack of disease occurrence. However, a lower estimated breeding value for ln(variance) was predictive for lower lesion scores after an avian pathogenic Escherichia coli inoculation and vice versa. In conclusion, this study shows that there is genetic variation in resilience indicators based on BW deviations in layer chickens, which opens up possibilities to improve resilience by means of selective breeding

Effect of aging and repeated intratracheal challenge on levels of cryptic and overt natural antibodies in poultry

Natural antibodies (NAb) have been divided in 2 classes: overt and cryptic. Overt NAb can be detected in unfractionated normal sera of nonimmunized mammals and chickens. Cryptic NAb as described in mammals need an in vitro physical or biochemical treatment to be detected, which may reflect their biochemical modification in situ during inflammation or infection. We studied the effect of concurrent primary, secondary, and tertiary intratracheal (i.t.) challenges with lipopolysaccharide (LPS) and human serum albumin on levels of 2 cryptic NAb [i.e., NAb binding actin (ACT), or thyroglobulin (THYRO)] and levels of an overt NAb binding keyhole limpet hemocyanin (KLH). In addition, effect of aging of the birds on levels of NAb was taken into account. Presence and changes on the level of the cryptic antibodies directed to ACT and THYRO were expected after the i.t. challenges with LPS (and human serum albumin). However, levels of NAb binding ACT were only significantly enhanced by LPS after primary challenge, whereas levels of NAb binding THYRO were not affected by primary nor secondary challenges but were significantly decreased by LPS after the third immunization. On the other hand, no changes in the levels of overt NAb binding KLH were expected, but levels of NAb binding KLH were significantly enhanced after the primary and secondary challenges with LPS. Levels of all three NAb increased with aging, but the different challenges performed at 3 moments during aging did not significantly or consistently affect levels of the overt nor cryptic NAb. Our results suggest that chickens might react by overt NAb as well as cryptic NAb to an infection or inflammation rather than by cryptic NAb only. The relation between various types of NAb and i.t. immunization with antigen and LPS and aging is discusse

Genomic Region Containing Toll-Like Receptor Genes Has a Major Impact on Total IgM Antibodies Including KLH-Binding IgM Natural Antibodies in Chickens

Natural antibodies (NAb) are antigen binding antibodies present in individuals without a previous exposure to this antigen. Keyhole limpet hemocyanin (KLH)-binding NAb levels were previously associated with survival in chickens. This suggests that selective breeding for KLH-binding NAb may increase survival by means of improved general disease resistance. Genome-wide association studies (GWAS) were performed to identify genes underlying genetic variation in NAb levels. The studied population consisted of 1,628 adolescent layer chickens with observations for titers of KLH-binding NAb of the isotypes IgM, IgA, IgG, the total KLH-binding (IgT) NAb titers, total antibody concentrations of the isotypes IgM, IgA, IgG, and the total antibodies concentration in plasma. GWAS were performed using 57,636 single-nucleotide polymorphisms (SNP). One chromosomal region on chromosome 4 was associated with KLH-binding IgT NAb, and total IgM concentration, and especially with KLH-binding IgM NAb. The region of interest was fine mapped by imputing the region of the study population to whole genome sequence, and subsequently performing an association study using the imputed sequence variants. 16 candidate genes were identified, of which FAM114A1, Toll-like receptor 1 family member B (TLR1B), TLR1A, Krüppel-like factor 3 (KLF3) showed the strongest associations. SNP located in coding regions of the candidate genes were checked for predicted changes in protein functioning. One SNP (at 69,965,939 base pairs) received the maximum impact score from two independent prediction tools, which makes this SNP the most likely causal variant. This SNP is located in TLR1A, which suggests a fundamental role of TLR1A on regulation of IgM levels (i.e., KLH-binding IgM NAb, and total IgM concentration), or B cells biology, or both. This study contributes to increased understanding of (genetic) regulation of KLH-binding NAb levels, and total antibody concentrations

Antigen-dependent effects of divergent selective breeding based on natural antibodies on specific humoral immune responses in chickens

NAb are defined as antigen binding antibodies present without a known previous exposure to this antigen. NAb are suggested to enhance specific antibody (SpAb) responses, but consequences of different NAb levels on immunization are largely unknown. Layer chickens were divergently selected and bred for keyhole limpet hemocyanin (KLH)-binding NAb titers, resulting in a High line and a Low line. In this study, we investigated: (1) the relation of NAb levels with SpAb titers; and (2) the effect of immunization on NAb titers. The 50 highest females of the High line and the 50 lowest females of the Low line of generation 2 were intramuscularly immunized at 33 weeks of age with 1 mL phosphate buffered saline (PBS) containing one of four treatments: (1) negative control (no antigen), (2) 500 μg KLH, (3) 100 μg avian tuberculin purified protein derivative of Mycobacterium avium (PPD), or (4) 250 μg human serum albumin (HuSA). IgM and IgG titers of NAb and SpAb in plasma were determined prior to immunization and weekly for 5 weeks post immunization by indirect ELISA. In addition, antibody affinity was investigated. No differences in SpAb and NAb response against KLH and PPD were observed as a consequence of different NAb titers, but increased and prolonged SpAb and NAb titer responses against HuSA were observed for the High line compared to the Low line. Different natural antibody titers did not impair SpAb dynamics and SpAb affinity. NAb titers were not, or for only short-term, affected by immunization. We show here that NAb may enhance SpAb responses, but that this effect is antigen-dependent. We hypothesize that NAb play a role in general disease resistance through enhancement of the humoral adaptive immune response

Effect of a TLR1A polymorphism on natural antibodies in poultry

The aim of the current study was to confirm and characterise the effect of a polymorphism in the Toll-like receptor 1 family member A (TLR1A) gene on levels of natural antibodies (NAb) in poultry. The study population consisted of a base population and 7 generations of birds from a High and Low selection line. Genotypes for a polymorphism in TLR1A were available for chickens in generations 5, 6 and 7. The TLR1A polymorphism had large and significant effects on total NAb levels (IgTotal) and IgM NAb, with full dominance of the TLR1A C allele. Significant (TLR1A*Generation) interactions were detected for IgM and IgTotal. The frequency of the TLR1A C allele was 0.45 in the base population (based on imputed genotypes), 0.66 and 0.04 in generation 7 of the High and Low line, respectively. Results confirm the major effect of this polymorphism in TLR1A on NAb levels suggest high sensitivity of the effect to environmental factors

Candidate gene for natural immune competence in chickens revealed through genome wide association study

Natural antibodies (NAb) are antibodies that are present in individuals without prior exposure to the antigen that they bind. In chickens, NAb have been associated with survival. Furthermore, genetic variation between chickens in NAb levels has been demonstrated. To improve our understanding of the genetic variation for NAb levels in chickens we performed a genome wide association study. A population of 1,628 White Leghorn chickens was investigated. Animals were phenotyped for NAb levels using the antigen keyhole limpet hemocyanin (KLH) as model. Animals were genotyped for 15,579 single nucleotide polymorphisms (SNP). Highly significant association between SNP and NAb levels was observed on chicken chromosome 4. A single SNP was identified as the most likely candidate for this association. Identification of this candidate SNP was based on further analysis with full genome sequence genotypes and on predicted consequences of associated SNP. This candidate SNP causes a phenylalanine to leucine amino acid change in one of the leucine rich repeats in toll-like receptor 1 family member A (TLR1A). Our results suggest an important role for TLR1A in relation to NAb levels in chickens. Further study should confirm the effect of the SNP in TLR1A on NAb levels and on the (natural) immune competence of chickens. Keywords: natural antibodies, toll-like receptor, TLR1

Genetic and Non-Genetic Inheritance of Natural Antibodies Binding Keyhole Limpet Hemocyanin in a Purebred Layer Chicken Line

Natural antibodies (NAb) are defined as antibodies present in individuals without known antigenic challenge. Levels of NAb binding keyhole limpet hemocyanin (KLH) in chickens were earlier shown to be heritable, and to be associated with survival. Selective breeding may thus provide a strategy to improve natural disease resistance. We phenotyped 3,689 white purebred laying chickens for KLH binding NAb of different isotypes around 16 weeks of age. Heritabilities of 0.12 for the titers of total antibodies (IgT), 0.14 for IgM, 0.10 for IgA, and 0.07 for IgG were estimated. We also estimated high, positive genetic, and moderate to high, positive phenotypic correlations of IgT, IgM, IgA, and IgG, suggesting that selective breeding for NAb can be done on all antibody isotypes simultaneously. In addition, a relatively substantial non-genetic maternal environmental effect of 0.06 was detected for IgM, which may reflect a transgenerational effect. This suggests that not only the genes of the mother, but also the maternal environment affects the immune system of the offspring. Breaking strength and early eggshell whiteness of the mother’s eggs were predictive for IgM levels in the offspring, and partly explained the observed maternal environmental effects. The present results confirm that NAb are heritable, however maternal effects should be taken into account

Localization and (semi-)quantification of fluorescent beads of 2 sizes in chickens over time after simultaneous intratracheal and cloacal administration

Environmental particles enter the chicken via several routes. Entry via the respiratory and cloacal routes likely activates immune responses. We studied the localization of simultaneous intratracheally and cloacally applied beads of 2 sizes in the chicken body in time, and when possible, semiquantified the amount of beads. Ten broiler hens, 3.5 wk of age, received 1.25 × 109 1.0-µm beads and 1.05 × 107 10-µm fluorescein isothiocyanate (green) labeled cloacally, and simultaneously the same number and same sizes of tetramethylrhodamine isothiocyanate (red) labeled beads intratracheally. The bursa of Fabricius, lung, liver, kidney, gallbladder, spleen, thymus, small intestine (upper ileum), cecum, intestinal luminal contents, aerated bones, feces, and blood, from 2 chickens per moment were sampled at 1 h, 6 h, 24 h, 48 h, and 1 wk after challenge and studied for the presence of beads using fluorescence microscopy. The highest amount of beads was found in organs closest to the application site after 1 h (i.e., the lungs for red beads, and the bursa for green beads). All tissue samples showed all 4 types of beads at all time moments, most of them within 1 h. Lower levels of beads were found in lungs and bursa after 6 h and in all other organs after 24 h, except for the kidneys where levels declined after 48 h. Surprisingly, beads were found in thymus tissue and only relatively few beads were found in the spleen. At 1 h, 1-µm intratracheally applied red beads were also found in the cecal luminal content and cecal tissue, but not in the small intestinal luminal content, suggesting that ceca are capable of excreting small particles entering the body via the respiratory route. The presence of nondegradable and nonimmunogenic beads of different sizes in all sampled organs throughout the whole chicken body for 7 d suggested potentially negative chronic health and welfare risks for the chicken of environmental particles

Genetic relations between natural antibodies binding keyhole limpet hemocyanin and production traits in a purebred layer chicken line

Natural antibodies (NAb) are an important component of the first line of immune defense. Selective breeding for enhanced NAb levels in chickens may improve general disease resistance. It is unknown what the consequences of selection for NAb will be on the productive performance of laying hens. In this paper we describe the genetic relations between NAb titers binding keyhole limpet hemocyanin at 19 wk age and production traits in a white purebred leghorn chicken line observed in several time periods. A linear animal model was used to estimate (co)variance components, heritabilities, and correlations. Negative genetic correlations were found between egg weight and NAb titers, and between egg breaking strength and NAb titers. Positive genetic correlations were found between the feed conversion ratio (consumed feed/egg mass produced) and NAb titers, and egg production and NAb titers. Negative phenotypic correlations were found between body weight and NAb titers, between egg weight and NAb titers, and between egg breaking strength and NAb titers. Positive phenotypic correlations were found between egg production and NAb titers, and feed conversion ratio and NAb titers. In general, phenotypic correlations were more often significant, but less pronounced than genetic correlations. Other production traits were not found to be significant related to NAb titers. These findings suggest that there is a genetic tradeoff between levels of immunity and some production traits, although the underlying mechanism(s) remain(s) unclear. The results suggest possible consequences for production efficiency as a result of selective breeding for improved general disease resistance by natural antibodies