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Background: Natural antibodies (NAb) are antibodies that are present in a healthy individual without requiring previous exposure to an exogenous antigen. Selection for high NAb levels might contribute to improved general disease resistance. Our aim was to analyse the genetic background of NAb based on a divergent selection experiment in poultry, and in particular the effect of a polymorphism in the TLR1A gene., Methods: The study population consisted of a base population from a commercial pure-bred elite white leghorn layer line and seven generations of birds from a High and Low selection line. Birds were selected for total KLH-binding NAb titer (IgTotal). An enzyme-linked immunosorbent assay was performed to determine NAb titers in blood plasma for IgTotal and the antibody isotypes IgM and IgG. NAb titers were available for 10,878 birds. Genotypes for a polymorphism in TLR1A were determined for chickens in generations 5, 6 and 7. The data were analysed using mixed linear animal models., Results: The heritability estimate for IgM was 0.30 and higher than that for IgG and IgTotal (0.12). Maternal environmental effects explained 2 to 3% of the phenotypic variation in NAb. Selection for IgTotal resulted in a genetic difference between the High and Low line of 2.4 titer points (5.1 genetic standard deviation) in generation 7. For IgM, the selection response was asymmetrical and higher in the Low than the High line. The frequency of the TLR1A C allele was 0.45 in the base population and 0.66 and 0.04 in generation 7 of the High and Low line, respectively. The TLR1A polymorphism had large and significant effects on IgTotal and IgM. Estimated genotypic effects suggest full dominance of the TLR1A C allele. Significant TLR1A by generation interactions were detected for IgM and IgTotal., Conclusions: The effect of a polymorphism in the TLR1A gene on IgTotal and IgM NAb was confirmed. Furthermore, we provide experimental verification of changes in allele frequencies at a major gene with dominant gene action on a quantitative trait that is subjected to mass selection. TLR1A by generation interactions indicate sensitivity to environmental factors., (© 2022. The Author(s).)

Keyhole limpet hemocyanin (KLH)-binding natural antibody (NAb) titers in chickens are heritable, and higher levels have previously been associated with a higher survival. This suggests that selective breeding for higher NAb levels might increase survival by means of improved general disease resistance. Chickens were divergently selected and bred for total NAb levels binding KLH at 16 weeks of age for six generations, resulting in a High NAb selection line and a Low NAb selection line. To for test differences in disease resistance, chickens were challenged with avian pathogenic Escherichia coli (APEC) in two separate experiments. Chickens at 8 days of age received one of four intratracheal inoculations of 0.2 mL phosphate buffered saline (PBS): 1) mock inoculate, 2) with 0.2 mL PBS containing 10 8.20  colony-forming units (CFU)/mL APEC, 3) with 0.2 mL PBS containing 10 6.64  CFU/mL APEC, and 4) with 0.2 mL PBS containing 10 7.55  CFU/mL APEC. Mortality was recorded during 7 days post inoculation. Overall, 50-60% reduced mortality was observed in the High line compared to the Low line for all APEC doses. In addition, morbidity was determined of the surviving chickens at 15 days of age. The High line had lower morbidity scores compared to the Low line. We conclude that selective breeding for high KLH-binding NAb levels at 16 weeks of age increase APEC resistance in early life. This study and previous studies support the hypothesis that KLH-binding NAb might be used as an indicator trait for to selective breed for general disease resistance in an antigen non-specific fashion., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Natural antibodies (NAb) are an important component of the innate immune system, and fight infections as a part of the first line defence. NAb are poly-reactive and can respond non-specifically to antigens. Therefore, NAb may be a key trait when evaluating an animal's potential natural disease resistance. Variation in NAb is caused by both genetic and environmental factors. In this study genetic parameters of NAb were estimated and a genome-wide association study (GWAS) was performed to gain further understanding on the genes that are responsible for the observed genetic variation of NAb in Canadian Holsteins., Results: In total, blood samples of 1327 cows from 64 farms were studied. NAb binding to keyhole limpet hemocyanin (KLH) were determined via indirect ELISA. Immunoglobulin (Ig) isotypes, IgG and IgM, were evaluated. From the sample population, 925 cows were genotyped for 45,187 markers and each individual marker was tested to detect genetic variation in NAb levels. The relationships among animals was accounted for with genomic relationship. Results show heritabilities of 0.27 ± 0.064 (IgG) and 0.31 ± 0.065 (IgM). In total, 23 SNPs were found to be associated with IgG, but no SNPs were associated with IgM (FDR p-value < 0.05). The significant SNPs were located on autosomal chromosomes 1, 20 and 21 of the cow genome. Functional annotation analysis of the positional candidate genes revealed two sets of genes with biologically relevant functions related to NAb. In one set, seven genes with crucial roles in the production of antibody in B cells were associated with the trafficking of vesicles inside the cells between organelles. In the second set, two genes among positional candidate genes were associated with isotype class-switching and somatic hypermutation of B cells., Conclusions: This study demonstrated the possibility of increasing NAb through selective breeding. In addition, the effects of two candidate pathways are proposed for further investigation of NAb production in Holsteins.

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., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

Levels of natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH) in layers were shown to be heritable and to be potential indicative parameters for survival. A proportion of NAb are directed to self-molecules, or slightly changed self-molecules (neo-epitopes), labeled as natural autoantibodies (NAAb). It is unknown whether the levels of NAAb are heritable and genetically correlated. In this paper, we estimated genetic parameters in plasma of healthy layers for IgM and IgG antibodies binding ovalbumin (OVA), myosin (MYO), cardiolipin (CAR), lysozyme (LYS), and the model antigen keyhole limpet hemocyanin (KLH). A linear animal model was used to estimate (co)variance components, heritabilities, and correlations. The estimates of heritabilities ranged from 0.10 to 0.17 for IgM, and 0.02 to 0.11 for IgG, respectively. For both IgM and IgG, high genetic correlations were observed between levels of NAAb binding autoantigens and NAb binding KLH, except for IgG binding KLH and LYS, for which a low genetic correlation was found. Low to moderate phenotypic correlations were found between NAAb and NAb. In addition, significant maternal environmental effects of 0.03, 0.07, and 0.04 were observed for IgM binding OVA, LYS, and KLH, respectively. Results from this study indicated that NAAb or NAb levels in plasma were heritable and could provide tools to identify the health status of birds., (© 2015 Poultry Science Association Inc.)

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.

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., (© 2015 Poultry Science Association Inc.)

To improve the health status (resilience) of dairy cows, levels of natural antibodies (NAb) might be useful. The objective of the present study was to compare levels and to estimate genetic parameters for NAb measured in milk and plasma samples. Titers of NAb IgM and IgG isotype-binding keyhole limpet hemocyanin of 2,919 cows, in both plasma and milk, were measured using ELISA. Analysis revealed that NAb levels in milk significantly increased with parity, whereas they remained constant in plasma. Moderate positive phenotypic correlations were found between NAb levels in milk and in plasma: 0.18 for IgG and 0.40 for IgM. This indicates that NAb from milk and plasma might reflect different aspects of dairy cow health status. However, high genetic correlations were found for NAb in milk and plasma: 0.81 for IgG and 0.79 for IgM. Heritabilities (SE in parentheses) for NAb measured in plasma [0.15 (0.05) for IgG and 0.25 (0.06) for IgM] were higher than heritabilities of NAb measured in milk [0.08 (0.03) for IgG and 0.23 (0.05) for IgM]. Our results indicate that NAb measured in milk and plasma are heritable and likely have a common genetic background, suggesting that NAb levels measured in milk might be useful for genetic improvement of disease resistance., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Because of a ban on the use of beak trimming in some European countries, feather pecking is becoming a substantial problem in the layer industry, both from animal welfare and economic points of view. The feather condition score (FCS) as a measure of feather damage has been shown to be closely related to feather pecking behavior in laying hens housed in groups. To obtain a better understanding of genetic and other biological mechanisms underlying feather pecking behavior, data on FCS of a population of 2,724 female offspring from crossing 50 male W1 and 907 female WB purebred lines were used. The offspring of 25 sires were beak-trimmed, and the offspring of another 25 sires were non-beak-trimmed. Titers of plasma natural antibody (NAb) isotypes IgM and IgG binding keyhole limpet hemocyanin at 24 wk of age were measured. Feather condition was scored at 53 wk of age. In the first part of the present study, we estimated genetic parameters for FCS with 2 variance components models: a traditional linear animal model and a model combining direct and associative genetic effects. In the second part of the present study, a trait-based analysis for FCS was conducted to investigate whether NAb isotype titers can explain variation in FCS among individuals, by fitting a linear mixed model. Though the estimated associative genetic variance was substantial, associative effects for FCS were not statistically significant in both populations (P = 0.09 in beak-trimmed birds, and P = 0.08 in non-beak-trimmed birds). This suggests an insufficient number of records on FCS. Individual's NAb isotypes titers did not show direct effect for FCS of itself, but individual's IgG titers showed a suggestive effect on the FCS of cage mates (associative effect) in beak-trimmed laying hens, which need further confirmation.

The objective of the present study was to estimate genetic parameters for natural antibody isotypes immunoglobulin (Ig) A, IgG1 and IgM titers binding the bacterial antigens lipopolysaccharide, peptidoglycan and the model antigen keyhole limpet hemocyanin in Dutch Holstein-Friesian cows (n = 1695). Further, this study included total natural antibody titers binding the antigens mentioned above, making no isotype distinction, as well as total natural antibody titers and natural antibody isotypes IgA, IgG1 and IgM binding lipoteichoic acid. The study showed that natural antibody isotype titers are heritable, ranging from 0.06 to 0.55, and that these heritabilities were generally higher than heritabilities for total natural antibody titers. Genetic correlations, the combinations of total natural antibody titers and natural antibody isotype titers, were nearly all positive and ranged from -0.23 to 0.99. Strong genetic correlations were found between IgA and IgM. Genetic correlations were substantially weaker when they involved an IgG1 titer, indicating that IgA and IgM have a common genetic basis, but that the genetic basis for IgG1 differs from that for IgA or IgM. Results from this study indicate that natural antibody isotype titers show the potential for effective genetic selection. Further, natural antibody isotypes may provide a better characterization of different elements of the immune response or immune competence. As such, natural antibody isotypes may enable more effective decisions when breeding programs start to include innate immune parameters., (© 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.)

Natural antibodies (NAb) are important humoral components of innate immunity. As the first line of defense, NAb provide protection against infection and support adaptive immunity. An earlier study indicated that serum levels of NAb isotypes IgM and IgG at a young age were predictive for survival in non-beak-trimmed purebred laying hens during the laying period. In the present study, genetic parameters of NAb isotypes were estimated and relationships between survival and NAb isotypes levels in crossbred laying hens were investigated. In total, 1,555 beak-trimmed and 1,169 non-beak-trimmed crossbred laying hens were used. Genetic parameters of IgM and IgG titers binding keyhole limpet hemocyanin at 24 wk of age were estimated with a linear animal model. The heritabilities of NAb isotypes IgG and IgM were 0.21 (SE = 0.04) and 0.26 (SE = 0.04), respectively. The genetic correlation between IgG and IgM isotypes was 0.43 (SE = 0.11). These results indicated that NAb isotype titers were heritable traits in the crossbred laying hens. Both NAb isotypes can be selected for simultaneously because the detected positive genetic correlation (0.43, SE = 0.11) between them is positive. Both row and level of the cage were indicated to be associated environmental factors for NAb isotype titers. Different from an earlier study with purebred hens, survival analysis showed no significant associations of survival with NAb isotype titers in beak-trimmed or non-beak-trimmed crossbred hens. Non-health-related causes of mortality, especially in birds with intact beaks, overruled the anticipated relationships between NAb isotype titers and survival.

In an earlier study, serum levels of natural antibody isotypes IgM- and IgG-binding keyhole limpet hemocyanin were found to be indicative for survival through the laying period of hens and therefore considered as promising traits for future implementation in breeding programs for higher survival of layers. In the present study, we first estimated the genetic parameters for the two isotypes at 20, 40 and 65 weeks of age (IgM20, IgM40 and IgM65; IgG20, IgG40 and IgG65). Pooled genetic parameters were estimated from the total population of 2504 hens from nine purebred layer lines, with line included in the model to account for admixture. Moderate heritabilities (0.14-0.44) indicated that selection for isotype titers is feasible, especially for IgM. Secondly, associations between 1022 SNP markers and the above-mentioned six immunological traits were estimated in 650 genotyped hens from the nine lines. The association study was performed across lines to detect markers that are closer to the QTL and have the same phase of association in the entire population. Forty-three significant associations between SNPs and isotype titers were detected. The SNPs of interleukins IL10 and IL19 were associated with both isotypes; SNPs of tripartite motif containing 33 (TRIM33) and IL6 showed significant association with IgG20 and IgM20 respectively; SNPs of heat shock protein 90kDa alpha (cytosolic), class B member 1 (HSP90AB1) were associated with IgG titers at older ages. Some detected SNPs were also reported associated with other immune and behavioral traits., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

Innate immunity plays an important role in preventing (barrier function) or combating infection (effector function). An important humoral component of innate immunity is formed by natural antibodies (NAb). The objectives of this study were to determine presence, variation among cows and repeatability within cows over time of total NAb titers directed to the pathogen-associated molecular patterns lipopolysaccharide, lipoteichoic acid (LTA) and peptidoglycan, and titers of NAb directed to the glycoprotein keyhole limpet hemocyanin in milk and plasma of individual cows. Furthermore in milk the antibody isotypes IgG1, IgG2, IgM and IgA binding LTA were analyzed. Ten milk and blood samples were obtained from each of 20 clinically healthy dairy cows from first to seventh parity during a period of 3 weeks. Total NAb binding lipopolysaccharide, LTA, peptidoglycan, and keyhole limpet hemocyanin were detected in milk and plasma, with titers considerably higher in plasma than in milk. Total NAb titers showed significant variation among cows, and repeatability within cows over time (ranging from 0.60 to 0.93). Titers of NAb in milk and plasma were positively correlated (correlation ranging from 0.69 to 0.91). Natural antibodies in milk binding LTA were of all 4 isotypes tested, although IgG2 was on average only present at low titers. All 4 isotypes in milk binding LTA also showed variation among cows, and repeatability within cows over time (ranging from 0.84 to 0.92). We conclude that NAb can be measured in a consistent and repeatable manner in bovine milk and blood plasma., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To identify possible relationships between survival and titers of natural antibody (NAb) isotypes in serum of laying hens, birds from 12 purebred layer lines of 2 commercial breeds, Rhode Island Red (n = 524) and White Leghorn (n = 538), were monitored for survival during one laying period (from 20 until 70 wk of age). Titers of NAb isotype IgM- and IgG-binding keyhole limpet hemocyanin (KLH) in serum were measured at 20, 40, and 65 wk of age, respectively. Overall, the titers of IgM and IgG binding KLH decreased with aging. At the same age, lines within breed showed significantly different titers of isotypes (P < 0.0001). Multivariable logistic regression analysis showed that NAb isotype IgM and IgG titers at 20 wk of age were associated with survival at 20 to 40 wk of age. In the R breed, odds ratios of 0.56 (P < 0.0001) for IgM and 0.72 (P = 0.02) for IgG were estimated; in the W breed, these were 0.74 (P < 0.01) and 0.99 (P = 0.95) for IgM and IgG, respectively. We conclude that titers of Nab isotypes, especially the IgM-binding KLH at 20 wk of age, are indicative for survival during the laying period. The higher the titers of NAb isotypes, the higher the probability of layers to survive.

Background: An association study between single nucleotide polymorphism markers (SNP) and (innate and adaptive) immune parameters but also feather condition score on the back, rump and belly of laying hens was performed. The immune parameters measured in blood samples were natural and acquired antibody titers and complement activity. Feather condition score as a measure of feather damage was determined, this parameter is closely related to feather pecking behavior in hens housed in groups.The aim of the study was to detect associations between genetic markers and immune parameters and feather condition score across nine lines of laying hens, focusing on the feather peckers as well as on the victims of feather pecking., Methods: A novel approach based on across-line analysis and testing of the SNP-by-line interaction was performed., Results: In total 59 significant associations between SNP and immune traits were detected. Previously identified QTL were confirmed and new associations of genes regulating immune function identified. The IL17A gene (chromosome 3) influences natural and acquired antibody titers and activation of classical and alternative complement pathways. The major histocompatibility complex on chromosome 16 showed significant association with natural and acquired antibody titers and classical complement activity. The IL12B and IRF1 genes on chromosome 13 were associated with natural antibody titers.The direct effect of the genotype of an individual on its feather condition and the associative effect of the genotype of the cage mates on the individual's feather condition were analyzed. The direct genetic effect can be described as the susceptibility to be pecked at, and the associative genetic effect as the propensity to perform feather pecking. Eleven significant associations were detected for the direct effect, and 81 for the associative effect. The serotonin receptor 2C (HTR2C) on chromosome 4 was highlighted in both analyses., Conclusions: Our results 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.

Background: Next generation sequencing technologies allow to obtain at low cost the genomic sequence information that currently lacks for most economically and ecologically important organisms. For the mallard duck genomic data is limited. The mallard is, besides a species of large agricultural and societal importance, also the focal species when it comes to long distance dispersal of Avian Influenza. For large scale identification of SNPs we performed Illumina sequencing of wild mallard DNA and compared our data with ongoing genome and EST sequencing of domesticated conspecifics. This is the first study of its kind for waterfowl., Results: More than one billion base pairs of sequence information were generated resulting in a 16× coverage of a reduced representation library of the mallard genome. Sequence reads were aligned to a draft domesticated duck reference genome and allowed for the detection of over 122,000 SNPs within our mallard sequence dataset. In addition, almost 62,000 nucleotide positions on the domesticated duck reference showed a different nucleotide compared to wild mallard. Approximately 20,000 SNPs identified within our data were shared with SNPs identified in the sequenced domestic duck or in EST sequencing projects. The shared SNPs were considered to be highly reliable and were used to benchmark non-shared SNPs for quality. Genotyping of a representative sample of 364 SNPs resulted in a SNP conversion rate of 99.7%. The correlation of the minor allele count and observed minor allele frequency in the SNP discovery pool was 0.72., Conclusion: We identified almost 150,000 SNPs in wild mallards that will likely yield good results in genotyping. Of these, ~101,000 SNPs were detected within our wild mallard sequences and ~49,000 were detected between wild and domesticated duck data. In the ~101,000 SNPs we found a subset of ~20,000 SNPs shared between wild mallards and the sequenced domesticated duck suggesting a low genetic divergence. Comparison of quality metrics between the total SNP set (122,000 + 62,000 = 184,000 SNPs) and the validated subset shows similar characteristics for both sets. This indicates that we have detected a large amount (~150,000) of accurately inferred mallard SNPs, which will benefit bird evolutionary studies, ecological studies (e.g. disentangling migratory connectivity) and industrial breeding programs.

Defense mechanisms of dairy cows against diseases partly rest on their naturally present disease resistance capacity. Natural antibodies (NAb) form a soluble part of the innate immune system, being defined as antibodies circulating in animals without prior intentional antigenic stimulation. Genetic selection on NAb titers in milk, therefore, might improve disease resistance. We estimated genetic parameters of NAb titers binding lipopolysaccharide, lipoteichoic acid (LTA), peptidoglycan, and keyhole limpet hemocyanin, and titers of the NAb isotypes IgG1, IgM, and IgA binding LTA in milk of Dutch Holstein-Friesian heifers. Natural antibody titers were measured in 1 milk sample from each of 1,939 Holstein-Friesian heifers and used for estimating genetic parameters of NAb titers. The data show that phenotypic variation exists among heifers in NAb titers binding lipopolysaccharide, LTA, peptidoglycan, and keyhole limpet hemocyanin, and the NAb isotypes IgG1, IgM, and IgA binding LTA in milk. High genetic correlations among NAb (ranging from 0.45 to 0.99) indicated a common genetic basis for the levels of different NAb in bovine milk. Intra-herd heritability estimates for NAb ranged from 0.10 to 0.53. The results indicated that NAb levels have potential for genetic selection., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in laying hens. For this purpose, the associations between 1022 single nucleotide polymorphism (SNP) markers and immune traits were studied in 583 hens from nine different layer lines. Immune traits were natural antibodies for keyhole limpet haemocyanin (KLH) and lipopolysaccharide (LPS) at 20, 40 and 65 weeks, acquired antibodies to the vaccinal virus of Newcastle disease at 20 weeks, and complement activity measured on sheep and bovine red blood cells at 20, 40 and 65 weeks. We adopted a novel approach based on across-line analysis and testing of the SNP-by-line interaction. Among lines, linkage disequilibrium is conserved at shorter distances than in individual lines; therefore, SNPs significantly associated with immune traits across lines are expected to be near the functional mutations. In the analysis, the SNPs 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. Ultimately, 59 significant associations between SNPs and immune traits were detected. Our results confirmed some previously identified QTL and identified new QTL potentially involved in the immune function. We found evidence for a role of IL17A (chromosome 3) in natural and acquired antibody titres and in the classical and alternative pathways of complement activation. The major histocompatibility genes on chromosome 16 showed significant association with natural and acquired antibody titres and classical complement activity. The IL12B gene on chromosome 13 was associated with natural antibody titres.

We present the results of a genome-wide scan to identify quantitative trait loci (QTL) that contribute to genetic variation in long-chain milk fatty acids. Milk-fat composition phenotypes were available on 1,905 Dutch Holstein-Friesian cows. A total of 849 cows and their 7 sires were genotyped for 1,341 single nucleotide polymorphisms across all Bos taurus autosomes (BTA). We detected significant QTL on BTA14, BTA15, and BTA16: for C18:1 cis-9, C18:1 cis-12, C18:2 cis-9,12, CLA cis-9,trans-11, C18:3 cis-9,12,15, the C18 index, the total index, total saturated fatty acids, total unsaturated fatty acids (UFA), and the ratio of saturated fatty acids:unsaturated fatty acids on BTA14; for C18:1 trans fatty acids on BTA15; and for the C18 and CLA indices on BTA16. The QTL explained 3 to 19% of the phenotypic variance. Suggestive QTL were found on 16 other chromosomes. The diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism on BTA14, which is known to influence fatty acid composition, most likely explains the QTL that was detected on BTA14.

Dietary fat may play a role in the aetiology of many chronic diseases. Milk and milk-derived foods contribute substantially to dietary fat, but have a fat composition that is not optimal for human health. We measured the fat composition of milk samples in 1918 Dutch Holstein Friesian cows in their first lactation and estimated genetic parameters for fatty acids. Substantial genetic variation in milk-fat composition was found: heritabilities were high for short- and medium-chain fatty acids (C4:0-C16:0) and moderate for long-chain fatty acids (saturated and unsaturated C18). We genotyped 1762 cows for the DGAT1 K232A polymorphism, which is known to affect milk-fat percentage, to study the effect of the polymorphism on milk-fat composition. We found that the DGAT1 K232A polymorphism has a clear influence on milk-fat composition. The DGAT1 allele that encodes lysine (K) at position 232 (232K) is associated with more saturated fat; a larger fraction of C16:0; and smaller fractions of C14:0, unsaturated C18 and conjugated linoleic acid (P < 0.001). We conclude that selective breeding can make a significant contribution to change the fat composition of cow's milk.

Pulmonary hypertension syndrome (PHS), also referred to as ascites syndrome, is a growth-related disorder of chickens frequently observed in fast-growing broilers with insufficient pulmonary vascular capacity at low temperature and/or at high altitude. A cross between two genetically different broiler dam lines that originated from the White Plymouth Rock breed was used to produce a three-generation population. This population was used for the detection and localization of quantitative trait loci (QTL) affecting PHS-related traits. Ten full-sib families consisting of 456 G2 birds were typed with 420 microsatellite markers covering 24 autosomal chromosomes. Phenotypic observations were collected on 4202 G3 birds and a full-sib across family regression interval mapping approach was used to identify QTL. There was statistical evidence for QTL on chicken chromosome 2 (GGA2), GGA4 and GGA6. Suggestive QTL were found on chromosomes 5, 8, 10, 27 and 28. The most significant QTL were located on GGA2 for right and total ventricular weight as percentage of body weight (%RV and %TV respectively). A related trait, the ratio of right ventricular weight as percentage to total ventricular weight (RATIO), reached the suggestive threshold on this chromosome. All three QTL effects identified on GGA2 had their maximum test statistic in the region flanked by markers MCW0185 and MCW0245 (335-421 cM).

The bacterial artificial clone-based physical map for chicken plays an important role in the integration of the consensus linkage map and the whole-genome shotgun sequence. It also provides a valuable resource for clone selection within applications such as fluorescent in situ hybridization and positional cloning. However, a substantial number of clone contigs have not yet been assigned to a chromosomal location or have an ambiguous chromosome assignment. In this study, 86 single nucleotide polymorphism markers derived from 86 clones were mapped on the genetic map. These markers added anchoring information for 56 clone contigs and 13 individual clones, covering a total of 57,145 clones.

In this report we describe the analysis of an advanced intercross line (AIL) to confirm the quantitative trait locus (QTL) regions found for fatness traits in a previous study. QTL analysis was performed on chromosomes 1, 3, 4, 15, 18, and 27. The AIL was created by random intercrossing in each generation from generation 2 (G(2)) onwards until generation 9 (G(9)) was reached. QTL for abdominal fat weight (AFW) and/or percentage abdominal fat (AF%) on chromosomes 1, 3 and 27 were confirmed in the G(9) population. In addition, evidence for QTL for body weight at the age of 5 (BW5) and 7 (BW7) weeks and for the percentage of intramuscular fat (IF%) were found on chromosomes 1, 3, 15, and 27. Significant evidence for QTL was detected on chromosome 1 for BW5 and BW7. Suggestive evidence was found on chromosome 1 for AFW, AF% and IF%, on chromosome 15 for BW5, and on chromosome 27 for AF% and IF%. Furthermore, evidence on the chromosome-wise level was found on chromosome 3 for AFW, AF%, and BW7 and on chromosome 27 for BW5. For chromosomes 4 and 18, test statistics did not exceed the significance threshold.

Linkage disequilibrium (LD) refers to the correlation among neighboring alleles, reflecting non-random patterns of association between alleles at (nearby) loci. A better understanding of LD in the porcine genome is of direct relevance for identification of genes and mutations with a certain effect on the traits of interest. Here, 215 SNPs in seven genomic regions were genotyped in individuals of three breeds. Pairwise linkage disequilibrium was calculated for all marker pairs. To estimate the extent of LD, all pairwise LD values were plotted against the distance between the markers. Based on SNP markers in four genomic regions analyzed in three panels from populations of Large White, Dutch Landrace, and Meishan origin, useful LD is estimated to extend for approximately 40 to 60 kb in the porcine genome.

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms (SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds (a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines--in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases.

The object of this research was to study the relationship between feather pecking and open-field activity in laying hens at two different ages. A population of 550 birds of a laying hen cross was subjected to an open-field test at 5 and 29 weeks of age and to a social feather pecking test at 6 and 30 weeks of age. Factor analysis was used to identify underlying factors for each test: pecking behavior (social test) and open-field activity (open-field test). In young birds, a positive phenotypic correlation of 0.24 was found between high open-field activity and high levels of pecking behavior (ground pecking, preening, gentle feather pecking, and wall pecking). In adults, a similar genetic correlation of 0.62 was found. At adult age, the factor pecking behavior consisted mainly of gentle and severe feather pecking. Between ages, a strong, negative genetic correlation of -0.65 was found between open-field activity at young age and pecking behavior at adult age, indicating that open-field activity levels in young birds may predict pecking behavior in adult hens., (Copyright 2004 Plenum Publishing Corporation)

The mapping resolution of the physical map for chicken Chromosome 4 (GGA4) was improved by a combination of radiation hybrid (RH) mapping and bacterial artificial chromosome (BAC) mapping. The ChickRH6 hybrid panel was used to construct an RH map of GGA4. Eleven microsatellites known to be located on GGA4 were included as anchors to the genetic linkage map for this chromosome. Based on the known conserved synteny between GGA4 and human Chromosomes 4 and X, sequences were identified for the orthologous chicken genes from these human chromosomes by BLAST analysis. These sequences were subsequently used for the development of STS markers to be typed on the RH panel. Using a logarithm of the odds (LOD) threshold of 5.0, nine linkage groups could be constructed which were aligned with the genetic linkage map of this chromosome. The resulting RH map consisted of the 11 microsatellite markers and 50 genes. To further increase the number of genes on the map and to provide additional anchor points for the physical BAC map of this chromosome, BAC clones were identified for 22 microsatellites and 99 genes. The combined RH and BAC mapping approach resulted in the mapping of 61 genes on GGA4 increasing the resolution of the chicken-human comparative map for this chromosome. This enhanced comparative mapping resolution enabled the identification of multiple rearrangements between GGA4 and human Chromosomes 4q and Xp.

The objective of the current study was to estimate genetic and phenotypic correlations among feather pecking (FP) behavior and stress response, immune response, and egg quality parameters. These traits have been measured in an F2 cross, coming from a cross between a high and a low FP line of laying hens. Heritabilities (h2) of stress response (32 wk), primary immune response to keyhole limpet hemocyanin (KLH) (36 wk) and Mycobacterium butyricum (39 wk), and egg quality parameters (35, 44, and 50 wk of age) were calculated. The h2 was 0.05 +/- 0.05 (SE) for stress response, 0.15 +/- 0.07 for antibody response to KLH, and 0.08 +/- 0.06 for antibody response to M. butyricum. The h2 for egg quality traits were in the range of 0.12 to 0.30. Significant phenotypic correlations were found between gentle FP in adult hens and stress response, egg weight at 44 and 50 wk, and egg deformation at 50 wk. Significant additive genetic correlations were found between severe FP in adult hens and antibody response to KLH (0.79 +/- 0.35), and between ground pecking in adult hens and egg deformation at 50 wk (0.63 +/- 0.26), and between ground pecking and eggshell strength at 35, 44, and 50 wk of age (-0.86 +/- 0.29, -0.81 +/- 0.20, -0.76 +/- 0.24, respectively).

Quantitative trait loci for BW at 4, 6, 8, 12, and 18 wk of age were detected in an experimental F2 cross of layers divergently selected for primary antibody response to SRBC. A negative phenotypic correlation between levels of antibody titers and BW, was reported earlier within founder lines. The entire experimental population was genotyped with 174 microsatellite markers equally distributed over the genome. Two genetic models were applied in the QTL analysis: a half-sib model and a line-cross model, both using the regression interval method. In the half-sib model, 3 QTL for BW at 4 wk of age on chromosomes GGA2, GGA3, and GGA9 were detected. For BW at 6 wk of age, 3 QTL were detected on GGA2, GGA3, and GGA6. For BW at 8 wk of age, a QTL was detected on GGA7, and for BW at 12 and 18 wk of age, a QTL was detected on GGAZ. With the line-cross analysis model, one QTL on GGA7 for BW at 4 wk of age was detected. Two QTL were detected on GGA3 and GGA7 for BW at 6 wk of age, and one on GGA3 was detected for BW for 8 and 12 wk of age. For BW at 18 wk of age, there were no QTL under the line-cross analysis model. The present data suggest that 1) a different set of genes affects the early and the late growth, and 2) genes selected to humoral immune responsiveness are different from genes underlying growth.

Line differences for open-field behavior in chickens have been observed, and it has been shown that this behavior has a genetic component. The aim of this study was to detect quantitative trait loci (QTL) involved in open-field behavior. For this purpose, open-field behavior was studied at 5 and 29 weeks of age in F(2) hens coming from an intercross between two commercial White Leghorn laying lines selected for egg production traits. Latencies, durations, and frequencies of general activity (sitting, standing, walking, and stepping), defecation, and vocalizations were recorded individually for each bird, and a factor score was calculated. All animals (F(0), F(1), and F(2)) were screened with 180 microsatellite markers. Regression interval mapping was applied using both a paternal half-sib analysis and a line-cross analysis method. For general activity at 5 weeks of age, a significant QTL was detected on GGA4 and a suggestive QTL on GGA2 under the line-cross model. For general activity at 29 weeks of age, a significant QTL was detected on GGA4 and two suggestive QTLs were detected on GGA1 and on GGA10, respectively, also using the line-cross analysis. The QTL on GGA4 at 5 weeks of age did not overlap with the QTL on GGA4 at 29 weeks of age. The current study indicated that open-field behavior in young chickens was regulated by QTL that differ from the QTL for open-field behavior in adult chickens.

A cross between 2 genetically different outcross broiler dam lines, originating from the White Plymouth Rock breed, was used to produce a large 3-generation broiler population. This population was used to detect and localize QTL affecting fatness in chicken. Twenty full-sib birds in generation 1 and 456 full-sib birds in generation 2 were typed for microsatellite markers, and phenotypic observations were collected for 3 groups of generation 3 birds (approximately 1,800 birds per group). Body weight, abdominal fat weight, and percentage abdominal fat was recorded at the age of 7, 9, and 10 wk. To study the presence of QTL, an across-family weighted regression interval mapping approach was used in a full-sib QTL analysis. Genotypes from 410 markers mapped on 25 chromosomes were available. For the 3 traits, 26 QTL were found for 18 regions on 12 chromosomes. Two genomewise significant QTL (P < 0.05) were detected, one for percentage abdominal fat at the age of 10 wk on chicken chromosome 1 at 241 cM (MCW0058 to MCW0101) with a test statistic of 2.75 and the other for BW at the age of 10 wk on chicken chromosome 13 at 9 cM (MCW0322 to MCW0110) with a test statistic of 2.77. Significance levels were obtained using the permutation test. Multiple suggestive QTL were found on chromosomes 1, 2, 4, 13, 15, and 18, whereas chromosomes 3, 7, 10, 11, 14, and 27 had a single suggestive QTL.

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.

Single nucleotide polymorphism markers are developed on SSC2, predominantly on the p-arm. Several studies reported a quantitative trait loci (QTL) for backfat thickness in this region. Single nucleotide polymorphisms were identified by comparative re-sequencing of polymerase chain reaction (PCR) products from a panel of eight individuals. The panel consisted of five Large Whites (each from a different Dutch breeding company), a Meishan, a Pietrain and a Wild Boar. In total, 67 different PCR products were sequenced and 301 SNPs were identified in 32,429 bp of consensus sequence, an average of one SNP in every 108 bp. After correction for sample size, this polymorphism rate corresponds to a heterozygosity value of one SNP in every 357 bp. For 63% of the SNPs, there was variation among the five Large Whites, and these SNPs are relevant for linkage and association studies in commercial populations. Comparing the Whites with other breeds revealed higher variation rates with: (i) Meishan, 89%; (ii) Pietrain, 69%; (iii) Wild Boar, 70%. Because many of the experimental populations to identify QTL are based on crosses between these breeds, these SNPs are relevant for the fine mapping of the QTL identified within these crosses.

Quantitative trait loci involved in the primary antibody response to keyhole lympet hemocyanin (KLH) and Mycobacterium butyricum were detected in two independent populations of laying hens. The first population was an F2 cross (H/L) of lines divergently selected for either high or low primary antibody responses to SRBC, and the second population was an F2 cross between 2 commercial layer lines displaying differences in feather pecking behavior (FP). Both populations were typed with microsatellite markers widely distributed over the genome with similar intervals between markers. Titers of antibodies binding KLH and M. butyricum were measured for all individuals by ELISA. Two genetic models were applied to detect QTL involved in the humoral immune response: a half-sib model and a line-cross model, both using the regression interval method. In the half-sib analysis, 2 QTL (on GGA14 and GGA27) were detected for the antibody response to KLH for the H/L population, and 2 QTL (on GGA14 and GGA18) were detected for the FP population. Only 1 QTL was detected for M. butyricum on GGA14 in the FP population using the half-sib analysis model. Two QTL were detected for the FP population on GGA2 and GGA3 using the line-cross analysis model. A QTL for the primary antibody response to KLH detected on GGA14 was validated in both populations under the half-sib analysis model. The present data suggest differences in the genetic regulation of antibody responses to two different T-cell dependent antigens.

The aim of this study is to detect quantitative trait loci (QTL) involved in the regulation of the primary and the secondary immune response to sheep red blood cells (SRBC) in a resource population using microsatellite DNA markers. The F2 resource population originates from a cross of two divergently selected lines for either high (H line) or low (L line) primary antibody response to SRBC. The F2 population consisted of six half-sib families, three families per each of reciprocal crosses. Total antibody titres to SRBC were determined by agglutination in serum from all birds. F2, F1 and F0 generations were genotyped for 170 microsatellite markers, using a whole-genome scan approach. The half-sib and the line-cross analyses were performed to determine QTL regions associated with regulation of the immune response. In the half-sib analysis, four QTL for SRBC primary response have been identified: on GGA3, GGA5, GGA16 and GGA23. No QTL was identified for SRBC secondary response under the half-sib model. In the line-cross analysis, three QTL were identified on GGA10, GGA16 and GGA27 for SRBC primary response and five QTL were identified on GGA6, GGA9, GGA15, GGA16 and GGA27 for SRBC secondary response. Subsequently, the family contribution of individual families to the QTL was analysed. The family with the largest contribution was genotyped with additional microsatellite markers in the QTL region on GGA5. The extended half-sib analysis with additional genotype information results in narrowing down the QTL region on GGA5.

Feather pecking (FP) is a major problem in cage and free-range housing systems. In free-range systems, FP is more difficult to control. It is not known why a victim is being pecked. It could be that a bird is genetically predisposed to be pecked. To study the genetics of FP behavior, a large F2 population of 630 hens was generated from a cross between two commercial laying lines differing in their propensity to feather peck. The traits measured at 6 and 30 wk of age were receiving gentle FP, receiving severe FP, and receiving aggressive pecking. In addition, receiving toe pecking (TP) was also measured at 30 wk of age. For receiving gentle FP at 6 wk of age, a significant QTL on GGA1 and three different suggestive QTL were identified on GGA2, GGA6, and GGA7, respectively. For receiving gentle FP at 30 wk of age, a suggestive QTL on GGA5 was detected. For receiving aggressive pecking, a suggestive QTL was detected on GGA3. For receiving TP, three suggestive QTL were detected on GGA1, GGA5, and GGA23, respectively. The different QTL detected for receiving gentle FP at different ages indicate that this trait is regulated by different genes in young and adult hens.

The physical and comparative map of GGA15 was improved by the construction of 9 BAC contigs around loci previously mapped on GGA15 by linkage analysis. In total, 240 BAC clones were isolated, covering 30-35% of GGA15, and 120 STS were developed (104 STS derived from BAC end sequences and 18 STS derived within genes). Seventeen chicken orthologues of human genes located on human Chr 22q11-q12 were directly mapped within BAC contigs of GGA15. Furthermore, the partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases and revealed matches to 26 genes, ESTs, and genomic clones located on HSA22q11-q12 and HSA12q24. These results provide a better alignment of GGA15 with the corresponding regions in human and mouse, and improve our knowledge of the evolution and dynamics of the vertebrate genome.

In the European Union, legislation concerning animal housing is becoming stricter because of animal welfare concerns. Feather pecking (FP) in large group housing systems is a major problem. It has been suggested that corticosterone (CORT) response to manual restraint as a measure for stress is associated with FP behavior. The aim of the current study was to identify QTL involved in FP behavior and stress response in laying hens. An F2 population of 630 hens was established from a cross between two commercial lines of laying hens differing in their propensity to feather peck. The behavioral traits, measured at 6 and 30 wk of age, were gentle FP, severe FP, and aggressive pecking. Toe pecking was measured at 30 wk of age and CORT response to manual restraint was measured at 32 wk. All animals were genotyped for 180 microsatellite markers. A QTL analysis was performed using a regression interval mapping method. At 6 wk of age, a suggestive QTL on GGA10 was detected for gentle FP. At 30 wk of age, suggestive QTL were detected on GGA1 and GGA2 for gentle FP. A significant QTL was detected on GGA2 for severe FP. At 32 wk of age, a suggestive QTL was detected on GGA18 for CORT response to manual restraint. In addition, a suggestive QTL was detected on GGA5 with possible maternal parent-of-origin effect for CORT response.

The objective of the current study was to estimate heritabilities (h2) of feather pecking and open-field response of laying hens at two different ages. An F2 cross, originating from a high and a low feather pecking line of laying hens, was used for the experiment. Each of the 630 birds of the F2 cross was subjected to an open-field test (individual, 10 min) at 5 and 29 wk of age and to a social feather pecking test (groups of five birds on wood shavings, 30 min) at 6 and 30 wk of age. Both tests were performed in a square open field (1.25 x 1.25 m). Behavior was recorded directly from a monitor. Heritabilities of feather pecking and open-field behaviors were calculated. In the open-field test at 5 wk of age, high h2 were found for most traits, ranging from 0.20 for the frequency of flying to 0.49 for number of steps. In the social test at 6 wk, gentle feather pecking (0.12) and ground pecking (0.13) were found to be heritable. When both tests were repeated at 29 and 30 wk of age, h2 estimates were lower for the open-field test, ranging from 0.10 for duration of sitting to 0.20 for latency to first step. In the social test, however, higher h2 estimates of 0.15 for gentle feather pecking and 0.30 for ground pecking were found compared with 6 wk of age. In conclusion, gentle feather pecking and open-field behaviors may be used in selection against feather pecking.

We studied the effects of Salmonella typhimurium lipopolysaccharide (LPS) on in vivo cytokine mRNA levels in chickens, and investigated whether these levels could be altered by different nutrients. Two hundred and forty chicks were assigned in a 2 x 4 factorial design of treatments. Factors were intravenous injection with S. typhimurium LPS, or saline (control), and four dietary fat sources: corn oil (CO), linseed oil (LO), menhaden oil and beef tallow (BT). Two hours after injection birds were killed and their spleens removed for RNA extraction. Quantitative real-time RT-PCR assays for mRNA of chicken IFN-gamma, IL-6, IL-8, IL-15, IL-18 and 28S rRNA were used to obtain the in vivo splenic cytokine profiles. Expression levels of IL-6, IL-8, IL-18 and IFN-gamma mRNA increased, but IL-15 mRNA decreased 2h after challenge with LPS compared with saline controls. In saline-injected control chickens, the dietary oil source did not affect the splenic mRNA level of any cytokine. In LPS challenged chickens IFN-gamma mRNA was significantly higher in the chickens fed the fish oil enriched diet compared with the LO, CO and BT enriched diets. The present data imply that avian IL-15 has, at least partially, a different function compared to its mammalian counterpart, and in addition, chicken innate immune responses might be affected differently by n-3 PUFA compared to mammals.

A comparative map was made of chicken chromosome 13 (GGA13) with a part of human chromosome 5 (HSA5). Microsatellite markers specific for GGA13 were used to screen the Wageningen chicken bacterial artificial chromosome (BAC) library. Selected BAC clones were end sequenced and 57 sequence tag site (STS) markers were designed for contig building. In total, 204 BAC clones were identified which resulted in a coverage of about 20% of GGA13. Identification of genes was performed by a bi-directional approach. The first approach starting with sequencing mapped chicken BAC subclones, where sequences were used to identify orthologous genes in human and mouse by a basic local alignment search tool (BLAST) database search. The second approach started with the identification of chicken orthologues of human genes in the HSA5q23-35 region. The chicken orthologous genes were subsequently mapped by fluorescent in situ hybridisation (FISH) and/or single neucleotide polymorphism typing. The total number of genes mapped on GGA13 is increased from 14 to a total of 20 genes. Genes mapped on GGA13 have their orthologues on HSA5q23-5q35 in human and on Mmu11, Mmu13 and Mmu18 in mouse.

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.

Comparative mapping between the human and chicken genomes has revealed a striking conservation of synteny between the genomes of these two species, but the results have been based on low-resolution comparative maps. To address this conserved synteny in much more detail, a high-resolution human-chicken comparative map was constructed from human chromosome 15. Mapping, sequencing, and ordering of specific chicken bacterial artificial chromosomes has improved the comparative map of chromosome 15 (Hsa15) and the homologous regions in chicken with almost 100 new genes and/or expressed sequence tags. A comparison of Hsa15 with chicken identified seven conserved chromosomal segments between the two species. In chicken, these were on chromosome 1 (Gga1; two segments), Gga5 (two segments), and Gga10 (three segments). Although four conserved segments were also observed between Hsa15 and mouse, only one of the underlying rearrangement breakpoints was located at the same position as in chicken, indicating that the rearrangements generating the other three breakpoints occurred after the divergence of the rodent and the primate lineages. A high-resolution comparison of Gga10 with Hsa15 identified 19 conserved blocks, indicating the presence of at least 16 intrachromosomal rearrangement breakpoints in the bird lineage after the separation of birds and mammals. These results improve our knowledge of the evolution and dynamics of the vertebrate genomes and will aid in the clarification of the mechanisms that underlie the differentiation between the vertebrate species.

Thirty-three androgenetic progeny groups of common carp were analysed using 11 microsatellite markers to (i) verify the homozygous status of the 566 androgenetic individuals, (ii) analyse the microsatellite allele segregation, and (iii) study the possible association of microsatellite alleles with phenotypic traits. In total, 92% of the androgenetic individuals proved to be homozygous at all 11 loci. Forty-three of the 47 heterozygous individuals were heterozygous at a single locus only. This heterozygosity was probably due to DNA fragments caused by UV irradiation of the eggs. although the maternal origin of the fragments could not be proved beyond doubt. Screening with 11 microsatellites also revealed two linkage groups, a segregation distortion at two microsatellite loci, and the possible association of some microsatellites with mass, length, stress-related plasma cortisol levels, and basal plasma glucose levels. The success of the linkage and association study could be explained by a low recombination frequency due to high chiasma interference. This would imply a relatively short genetic map for common carp.

Effects of dietary polyunsaturated fatty acids (PUFA) on immune responses in poultry have been reported. However, effects on the underlying mechanisms, such as the role of cytokines, have not been documented because the necessary tools were lacking. Recently, primer sets for chicken interleukin (IL)-1beta, IL-2, interferon-gamma (IFN-gamma), myelomonocytic growth factor (MGF), and transforming growth factor (TGF)-beta2 have become available. Therefore, in the present study we first examined the in vivo effects of an inflammatory challenge with Salmonella typhimurium lipopolysaccharide (LPS) on cytokine profiles in growing laying-type chicks. Second, we examined whether dietary fat sources affected the observed cytokine profiles. Two hundred forty chicks were assigned in a 2 x 4 factorial design of treatments, with injection with LPS or saline and dietary fat source as factors. Factors were i.v. injection with S. typhimurium LPS or saline (control) and four dietary fat sources: corn oil, linseed oil, menhaden oil, and tallow. Two hours after injection, birds were killed, and their spleens were removed for RNA extraction. Reverse transcription polymerase chain reactions with primer sets for chicken IL-1beta, IL-2, IFN-gamma, MGF, TGF-beta2, and beta-actin were performed with RNA samples pooled by pen. The expression of cytokine mRNA was expressed relative to the level of beta-actin mRNA. Interleukin-1 (P < 0.001), MGF (P < 0.0001), IL-2 (P < 0.001), and IFN-gamma (P < 0.001) mRNA expressions were enhanced by challenge with LPS. Immunization treatment had no effect on TGF-beta2 or beta-actin expression. Dietary treatment did not affect mRNA expression of IL-1, MGF, IFN-gamma, TGF-beta2, or beta-actin. Interleukin-2 expression in LPS-injected birds that were fed the fish-oil-enriched diet was enhanced (P = 0.05). The present study indicates that in vivo effects of immune challenge on cytokine mRNA expression can be measured in poultry. The observation that mRNA level of IL-2, but not the mRNA levels of IFN-gamma or MGF, is enhanced by dietary fish oil at 2 h suggests that dietary PUFA at this moment initially affected naïve T lymphocytes.

We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies.

A bovine mammary gland cDNA-library was used to characterize and map genes expressed during lactation. Fifty cDNA clones selected by differential hybridization were sequenced from both ends, and sequences were examined for similarities with database sequences. For 34 of the transcripts, the sequences showed more than 80% similarity to previously characterized genes or expressed sequence tags (ESTs). Twenty cDNAs that could be of interest as candidate genes for milk production are selected for genetic or chromosomal mapping. Twenty-three out of the 39 designed primer pairs representing 16 cDNA clones amplified the expected fragments and were used for subsequent fluorescent single-strand conformation polymorphism analysis (F-SSCP) in the International Bovine Reference Panel families (IBRP). Ten polymorphic loci could be identified and used to genotype the IBRP animals, and nine of them were subsequently genetically mapped on nine chromosomes. In addition, eight loci from the 16 cDNA clones could be mapped by somatic cell hybrids, bringing the total number of mapped genes to 16, one of which was mapped genetically as well as physically. The mapped mammary gland ESTs are potentially useful for cloning economic trait loci by a positional candidate gene cloning approach.