Divergent selection for natural antibodies in poultry in the presence of a major gene.

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.

Genetic parameters of colostrum and calf serum antibodies in Swedish dairy cattle.

BACKGROUND: A sufficient IgG content in the colostrum is essential for the newborn calf, as it provides passive immunity which substantially affects the probability of survival during rearing. Failure of passive transfer (FPT) occurs when a calf does not absorb enough antibodies from the colostrum and is defined by an IgG concentration in calf serum lower than 10 g/L. Apart from delayed access to colostrum, FPT can be due to a low production of IgG in the mother or poor IgG absorption by the calf. The aim of this study was to estimate the genetic background of antibody levels and indicator traits for antibodies in the colostrum and calf serum, and their correlation with milk production. RESULTS: Colostrum data were available for 1340 dairy cows with at least one calving and calf serum data were available for 886 calves from these cows. Indicator traits for antibody concentrations were estimated using refractometry (a digital Brix refractometer for colostrum and an optical refractometer for serum), and enzyme-linked immunosorbent assays (ELISA) were used to determine the levels of total IgG and natural antibodies (NAb) of various antibody isotypes in the colostrum and calf serum. Colostrum traits had heritabilities ranging from 0.16 to 0.31 with repeatabilities ranging from 0.21 to 0.55. Brix percentages had positive genetic correlations with all colostrum antibody traits including total IgG (0.68). Calf serum antibody concentrations had heritabilities ranging from 0.25 to 0.59, with a significant maternal effect accounting for 17 to 27% of the variance. When later in life calves produced their first lactation, the lactation average somatic cell score was found to be negatively correlated with NAb levels in calf serum. CONCLUSIONS: Our results suggest that antibody levels in the colostrum and calf serum can be increased by means of selection.

Genome-wide association study identifies loci influencing natural antibody titers in milk of Dutch Holstein-Friesian cattle.

Natural antibodies (NAb) are produced without any antigenic stimulation as a part of the innate immune system and provide a first line of defense against pathogens. Hence, they may be a useful trait when estimating an animal's potential immune competence and in selection for disease resistance. The aim of this study was to identify genomic regions associated with different NAb traits in milk and potentially describe candidate genes. Milk samples from 1,695 first-lactation Holstein Friesian cows with titer measurements for keyhole limpet hemocyanin, lipopolysaccharide, lipoteichoic acid, and peptidoglycan-binding total NAb and isotypes IgG1, IgM, and IgA were used. Genome-wide association study analyses were performed using imputed 777K SNP genotypes, accounting for relationships using pedigree information. Functional enrichment analysis was performed on the significantly associated genomic regions to look for candidate genes. For IgM NAb, significant associations (false discovery rate <0.05) were found on Bos taurus autosome (BTA) 17, 18, and 21 with candidate genes related to immunoglobulin structure and early B cell development. For IgG1, associations were found on BTA3, and we confirmed a quantitative trait loci on BTA21 previously reported for IgG NAb in serum. Our results provide new insights into the regulation of milk NAb that will help unravel the complex relationship between milk immunoglobulins and disease resistance in dairy cattle.

Breeding for high specific immune reactivity affects sensitivity to the environment.

Lipopolysaccharides (LPS) are present in high levels in the air of chicken houses and likely have immune-modulating effects. In this study, layers from the 30th generation of a divergent selection experiment for humoral immune reactivity to subcutaneously administered sheep red blood cells were concurrently intratracheally challenged with human serum albumin (HuSA) and LPS at 7 and 12 wk of age following a crossing over design. Chickens selected for high humoral immune reactivity (H line) showed higher specific antibody responses to intratracheally administered HuSA and a higher level of natural antibody binding keyhole limpet hemocyanin compared with chickens genetically selected for low humoral immune reactivity (L line), and were also more sensitive to immune modulation by LPS at 7 wk of age. Body weight gain was negatively affected by LPS at 7 wk of age in the L line, but after 12 wk of age in the H line. Egg production was lower and delayed in the H line. We conclude that these chicken selection lines differ in sensitivity to the environment (LPS), and consequently may therefore also react differently to infection, vaccinations, and other immune responses. In addition, selection for immune responsiveness affected growth and egg lay.

Transgenerational Effects of Maternal Immune Activation on Specific Antibody Responses in Layer Chickens.

Activation of the maternal immune system may affect innate and adaptive immune responses in the next generation and may therefore have implications for vaccine efficacy and dietary immune modulation by feed additives. However, transgenerational effects on immune responses in chickens have been investigated to a limited extend. The present study investigated effects of intratracheal (i.t) specific and aspecific immune activation of laying hens on specific antibody production in the next generation. In two experiments laying hens received intratracheally an immune stimulus with human serum albumin (HuSA) or lipopolysaccharide (LPS). In experiment 1, hatchlings of the immune activated hens were at 4 weeks i.t. immunized with HuSA or HuSA+LPS. Maternal immune activation with LPS increased HuSA specific IgY and IgM responses in offspring. These results suggest a transgenerational effect of the maternal immune system on the specific antibody response in the next generation. In experiment 2 hatchlings received either ß-glucan-enriched feed or control feed and were i.t. immunized with HuSA. Maternal immune activation with LPS decreased IgY anti-HuSA responses after HuSA immunization within hatchlings that received ß-glucan enriched feed. The results of Experiment 2 suggest a transgenerational link between the innate immune system of mother and specific antibody responses in offspring. Despite variabilities in the outcomes of the two experiments, the observations of both suggest a link between the maternal innate immune system and the immune system of the offspring. Furthermore, our results may imply that maternal activation of the innate immune system can influence immune modulating dietary interventions and vaccine strategies in the next generation.

Association of MHCY genotypes in lines of chickens divergently selected for high or low antibody response to sheep red blood cells.

The chicken MHCY region contains members of several gene families including a family of highly polymorphic MHC class I genes that are structurally distinct from their classical class I gene counterparts. Genetic variability at MHCY could impart variability in immune responses, but robust tests for whether or not this occurs have been lacking. Here we defined the MHCY genotypes present in 2 sets of chicken lines selected for high or low antibody response, the Virginia Tech (VT) HAS and LAS, and the Wageningen University (WU) HA and LA lines. Both sets were developed under long-term bidirectional selection for differences in antibody responses following immunization with the experimental antigen sheep red blood cells. Lines in which selection was relaxed (VT HAR and LAR) or lacking (WU C) provided controls. We looked for evidence of association between MHCY genotypes and antibody titers. Chickens were typed for MHCY using a recently developed method based on a multilocus short tandem repeat sequence found across MHCY haplotypes. Five MHCY haplotypes were found segregating in the VT HAS and LAS lines. One haplotype was present only in HAS chickens, and another was present only in LAS chickens with distribution of the remaining 3 haplotypes differing significantly between the lines. In the WU HA and LA lines, there was a similar MHCY asymmetry. The control populations lacked similar asymmetries. These observations support the likelihood of MHCY genetics affecting heritable antibody responses and provide a basis for further investigations into the role of MHCY region genes in guiding immune responses in chickens.

Respiratory health of broilers following chronic exposure to airborne endotoxin.

In and around poultry farms, high concentrations of endotoxins are found that have a negative impact on the health of farmers and local residents. However, little is known about the effects of chronic exposure to endotoxins on the health of poultry. The aim of this study was to identify effects of chronic exposure to airborne endotoxins (E. coli LPS) on the immune system, respiratory tract, disease susceptibility and welfare of broilers. Effects of high (HE) and low endotoxin (LE) concentrations on natural antibody titers (NAb), performance and behavior of broilers were determined. After treatment with a respiratory virus infection, infectious bronchitis virus (IBV), mRNA expression of cytokines and Toll-like receptor (TLR) 4 in the lung, tracheal ciliary activity and lesions in the respiratory tract were determined. Endotoxin affected the immune system and respiratory tract, where HE broilers tended to have lower IgM NAb binding Phosphorylcholine-conjugated to Bovine Serum Albumin, and higher interferon (IFN)-α mRNA expression and more lesions in the nasal tissue compared to LE broilers. Furthermore, HE broilers had higher TLR4 mRNA expression compared to LE broilers. However, endotoxin did not affect NAb levels binding Keyhole Limpet Hemocyanin, IFN-ß and interleukin-10 mRNA expression, IBV replication or lesions in the lung and trachea. HE and LE broilers further had similar body weight, but HE broilers showed numerically more passive behavior compared to LE broilers. In conclusion, chronic exposure to high airborne endotoxin concentrations affects components of the immune system and respiratory tract in broilers and could therefore influence disease susceptibility.

Gut microbiota of homing pigeons shows summer-winter variation under constant diet indicating a substantial effect of temperature.

BACKGROUND: Gut microbiotas play a pivotal role in host physiology and behaviour, and may affect host life-history traits such as seasonal variation in host phenotypic state. Generally, seasonal gut microbiota variation is attributed to seasonal diet variation. However, seasonal temperature and day length variation may also drive gut microbiota variation. We investigated summer-winter differences in the gut bacterial community (GBC) in 14 homing pigeons living outdoors under a constant diet by collecting cloacal swabs in both seasons during two years. Because temperature effects may be mediated by host metabolism, we determined basal metabolic rate (BMR) and body mass. Immune competence is influenced by day length and has a close relationship with the GBC, and it may thus be a link between day length and gut microbiota. Therefore, we measured seven innate immune indices. We expected the GBC to show summer-winter differences and to correlate with metabolism and immune indices. RESULTS: BMR, body mass, and two immune indices varied seasonally, other host factors did not. The GBC showed differences between seasons and sexes, and correlated with metabolism and immune indices. The most abundant genus (Lachnoclostridium 12, 12%) and associated higher taxa, were more abundant in winter, though not significantly at the phylum level, Firmicutes. Bacteroidetes were more abundant in summer. The Firmicutes:Bacteroidetes ratio tended to be higher in winter. The KEGG ortholog functions for fatty acid biosynthesis and linoleic acid metabolism (PICRUSt2) had increased abundances in winter. CONCLUSIONS: The GBC of homing pigeons varied seasonally, even under a constant diet. The correlations between immune indices and the GBC did not involve consistently specific immune indices and included only one of the two immune indices that showed seasonal differences, suggesting that immune competence may be an unlikely link between day length and the GBC. The correlations between the GBC and metabolism indices, the higher Firmicutes:Bacteroidetes ratio in winter, and the resemblance of the summer-winter differences in the GBC with the general temperature effects on the GBC in the literature, suggest that temperature partly drove the summer-winter differences in the GBC in homing pigeons.

Transfer of natural auto-antibodies via egg yolk in chickens divergently selected for natural antibodies binding keyhole limpet hemocyanin.

Barcodes of natural auto-antibody (NAAb) profiles based on staining intensity of isotypes binding numbers of self-(tissue) antigen fragments were suggested as parameters for immune diversity, and related to genetic background and health status in man, rodents and poultry. Here, hens, eggs and hatchlings from chicken lines divergently selected and bred for high (H line) or low (L line) total natural antibodies (NAb) levels in plasma binding keyhole limpet hemocyanin (KLH) at 16 weeks of age were tested for their NAAb repertoire binding chicken liver homogenate (CLH) fragments using quantitative Western immunoblotting. The aims of this study were 1. to detect line differences between the H and L line adult hens, eggs and hatchlings for the IgM and IgG isotypes binding CLH fragments, 2. study the presence of NAAb of both isotypes in yolk and albumen, as well as in hatchlings to detect a maternal NAAb transfer route via the egg to the hatchling, and 3. study whether new self-antigen binding isotypes and idiotypes are present in the hatchling. NAAb binding CLH fragments were found in plasma of adult hens (both IgM and IgG), in yolk (IgG only), and hatchlings (mostly IgG, but low levels of IgM). Auto-profiles of IgM showed homogeneity, while IgG profiles were heterogenic between individual hens and individual hatchlings. Significant higher levels as indicated by staining intensity and number of stained CLH fragments were found in plasma of hens genetically selected for high levels of NAb binding KLH. Lines could be clustered based on their auto-profiles indicating that profiles of self-binding IgM and IgG antibodies are genetically based. Visual comparison, clustering and correlation of hens and their hatchlings showed similarities for the IgG, but not the IgM isotype, indicating maternal transfer of IgG NAAb via the yolk. The IgM profile in the hatchlings on the other hand might represent neonatal self-binding antibody formation. As a consequence, hatchlings initially depend for self-binding antibodies on maternal IgG provision during early life.

Current Understanding of Natural Antibodies and Exploring the Possibilities of Modulation Using Veterinary Models. A Review.

Natural antibodies (NAb) are defined as germline encoded immunoglobulins found in individuals without (known) prior antigenic experience. NAb bind exogenous (e.g., bacterial) and self-components and have been found in every vertebrate species tested. NAb likely act as a first-line immune defense against infections. A large part of NAb, so called natural autoantibodies (NAAb) bind to and clear (self) neo-epitopes, apoptotic, and necrotic cells. Such self-binding antibodies cannot, however, be considered as pathogenic autoantibodies in the classical sense. IgM and IgG NAb and NAAb and their implications in health and disease are relatively well-described in humans and mice. NAb are present in veterinary (and wildlife) species, but their relation with diseases and disorders in veterinary species are much less known. Also, there is little known of IgA NAb. IgA is the most abundant immunoglobulin with essential pro-inflammatory and homeostatic properties urging for more research on the importance of IgA NAb. Since NAb in humans were indicated to fulfill important functions in health and disease, their role in health of veterinary species should be investigated more often. Furthermore, it is unknown whether levels of NAb-isotypes and/or idiotypes can and should be modulated. Veterinary species as models of choice fill in a niche between mice and (non-human) primates, and the study of NAb in veterinary species may provide valuable new insights that will likely improve health management. Below, examples of the involvement of NAb in several diseases in mostly humans are shown. Possibilities of intravenous immunoglobulin administration, targeted immunotherapy, immunization, diet, and genetic modulation are discussed, all of which could be well-studied using animal models. Arguments are given why veterinary immunology should obtain inspiration from human studies and why human immunology would benefit from veterinary models. Within the One Health concept, findings from veterinary (and wildlife) studies can be related to human studies and vice versa so that both fields will mutually benefit. This will lead to a better understanding of NAb: their origin, activation mechanisms, and their implications in health and disease, and will lead to novel health management strategies for both human and veterinary species.

Effects of Early Nutrition and Sanitary Conditions on Oral Tolerance and Antibody Responses in Broiler Chickens.

Greater antigenic exposure might accelerate activation and maturation of the humoral immune system. After hatch, commercial broiler chickens can have early (EN) or delayed (DN) access to nutrition, up to 72 h after hatch. The immune system of EN versus DN broilers is likely more exposed to antigens after hatch. This might contribute to activation and maturation of the immune system, but might also influence the development of oral tolerance, thereby altering later life antibody responses. We studied antibody (IgM, IgY, IgA) responses between 21 and 42 d of age in fast-growing EN and DN broilers, kept under low (LSC) or high sanitary conditions (HSC). In a first experiment (n = 51 broilers), we tested whether early oral exposure to bovine serum albumin (BSA) affected later life antibody responses towards BSA and a novel antigen-rabbit γ-globulin (RGG), under HSC. In a second experiment, a total of 480 EN and DN broilers were housed under either LSC or HSC, and we studied antibody responses against both BSA and RGG (n = 48 broilers per treatment) and growth performance. Broilers kept under LSC versus HSC, had higher antibody levels and their growth performance was severely depressed. Interactions between feeding strategy (EN versus DN) and sanitary conditions, or main effects of feeding strategy, on natural and specific antibody levels, and growth performance were not observed. Levels of IgA were elevated in EN versus DN broilers, in experiment I and in batch 2 of experiment II, but not in the other batches of experiment II. We concluded that EN versus DN contributes minimally to the regulation of antibody responses, irrespective of antigenic pressure in the rearing environment.

Maternal Transfer of Natural (Auto-) Antibodies in Chickens.

The presence and relative levels (titers) of IgM and IgG natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH), and natural (auto-) antibodies (N(A)Ab) binding salmon double-stranded DNA (dsDNA), (oxidated-) phosphatidyl (phosphoryl) choline-conjugated bovine serum albumin (PC-BSA), PC-conjugated ovalbumin (PC-OVA), and OVA, respectively, were studied in adult hen plasma, egg yolk, egg albumen, plasma of their hatchlings, and in 8-day-old chick plasma. Birds and eggs were from 2 lines divergently selected for high or low NAb levels binding KLH. This study aimed to determine 1) correlated phenotypic responses of selection for NAb to KLH, 2) transfer of maternal NAb and N(A)Ab via egg compartments, 3) levels of likely maternal NAb and N(A)Ab in hatchlings and 8-day-old chicks, and 4) whether a composite trait: IgM anti-PC-BSA/IgG anti-dsDNA ratio in the compartments could be used as a parameter for health or immune status. NAb and N(A)Ab to all tested antigens were found in adult hens, but low or no levels were found for IgM in yolk and IgG in albumen. Depending on the antigen, NAb and N(A)Ab were found in hatchlings and day 8 birds. Divergent selection and breeding based on NAb binding KLH affected antibody titers of almost all antigens in almost all compartments, in a similar way. Maternal transfer of NAb and N(A)Ab from the adult hen to offspring was via specific routes for specific antigens and isotypes, especially for IgG as suggested by cluster analyses and significant correlations. There was little indication of production of new NAb and N(A)Ab to the studied antigens in either the egg compartments or the hatchlings. A composite trait of IgM PC-BSA/IgG dsDNA ratio was as yet not indicative for immune status, as no significant differences were found between the lines for all compartments. In conclusion, hens provide neonatal chickens with natural (self-) binding IgG antibodies that have been proposed to perform homeostatic functions during the period in which neonates do not produce these antibodies themselves.

Does paternal immunocompetence affect offspring vulnerability to maternal androgens? A study in domestic chickens.

Exposure of yolk androgens can positively stimulate chick growth and competitive ability, but may negatively affect immunity. It has been hypothesized that only chicks from immunologically superior fathers can bear the cost of prenatal exposure to high androgen levels. To test this hypothesis, we paired roosters from two selection lines, one up- and one down-selected for natural antibodies (NAbs), with hens from a control line. We measured yolk testosterone and androstenedione levels, and we injected the treatment group of eggs of each female with testosterone suspended in sesame oil and the control group with sesame oil only. We then measured hatching success and growth, and characterized the humoral and cellular immune responses using three different challenges: a phyto-hemagglutinin, a lipopolysaccharide and a sheep red blood cell challenge. We found that the hatching success, body mass, initial levels of natural antibodies and the chicks' immunological responses to the three different challenges and development were affected neither by paternal immunocompetence nor by treatment. These results do not support the hypothesis that chicks from low-NAb line fathers are more sensitive to testosterone exposure during embryonic development than chicks from high-NAb line fathers.

Relationship between inflammatory biomarkers and oxidative stress with uterine health in dairy cows with different dry period lengths.

Earlier studies indicated that the inflammatory status of dairy cows in early lactation could not be fully explained by the negative energy balance (NEB) at that moment. The objective of the present study was to determine relationships between inflammatory biomarkers and oxidative stress with uterine health in dairy cows after different dry period lengths. Holstein-Friesian dairy cows were assigned to one of three dry period lengths (0-, 30-, or 60-d) and one of two early lactation rations (glucogenic or lipogenic ration). Cows were fed either a glucogenic or lipogenic ration from 10-d before the expected calving date. Part of the cows which were planned for a 0-d dry period dried themselves off and were attributed to a new group (0 → 30-d dry period), which resulted in total in four dry period groups. Blood was collected (N = 110 cows) in weeks -3, -2, -1, 1, 2, and 4 relative to calving to determine biomarkers for inflammation, liver function, and oxidative stress. Uterine health status (UHS) was monitored by scoring vaginal discharge (VD) based on a 4-point scoring system (0, 1, 2, or 3) in weeks 2 and 3 after calving. Cows were classified as having a healthy uterine environment (HU, VD score = 0 or 1 in both weeks 2 and 3), nonrecovering uterine environment (NRU, VD score = 2 or 3 in week 3), or a recovering uterine environment (RU, VD score = 2 or 3 in week 2 and VD score= 0 or 1 in week 3). Independent of dry period length, cows with NRU had higher plasma haptoglobin (P = 0.05) and lower paraoxonase levels (P < 0.01) in the first 4 weeks after calving and lower liver functionality index (P < 0.01) compared with cows with HU. Cows with NRU had lower plasma albumin (P = 0.02) and creatinine (P = 0.02) compared with cows with a RU, but not compared with cows with HU. Independent of UHS, cows with a 0 → 30-d dry period had higher bilirubin levels compared with cows with 0-, 30-, or 60-d dry period (P < 0.01). Cows with RU and fed a lipogenic ration had higher levels of albumin in plasma compared with cows with NRU and fed a lipogenic ration (P < 0.01). In conclusion, uterine health was related to biomarkers for inflammation (haptoglobin and albumin) and paraoxonase in dairy cows in early lactation. Cows which were planned for a 0-d dry period, but dried themselves off (0 → 30-d dry period group) had higher bilirubin levels, which was possibly related to a more severe NEB in these cows. Inflammatory biomarkers in dairy cows in early lactation were related to uterine health in this period.

Correlated effects of selection for immunity in White Leghorn chicken lines on natural antibodies and specific antibody responses to KLH and M. butyricum.

BACKGROUND: The effect of selection for three general immune response traits on primary antibody responses (Ab) to Mycobacterium butyricum or keyhole limpet hemocyanin (KLH) was studied in four experimental lines of White Leghorn chicken. Birds underwent 12 generations of selection for one of three different general immune criteria; high antibody response to Newcastle disease virus 3 weeks after vaccination (ND3), high cell-mediated immune response, using the wing web response to phytohemglutinin (PHA) and high phagocytic activity, measured as carbone clearance (CC). Line ND3-L was selected on ND3, line PHA-L was selected on PHA, and line CC-L on CC, but all lines were measured for all three traits. The fourth line was a contemporary random bred control maintained throughout the selection experiment. Principal component analysis was used to distinguish clusters based on the overall set of immune measures. RESULTS: In the KLH immunised group, no differences were present between lines for natural antibodies binding to KLH and LPS, and, lines ND3-L and PHA-L had higher titers to LTA and anti-Gal titers measured before the immunisation protocol. The measure of ND3 was correlated positively with LPS titers measured post KLH immunisation and with the difference between LPS titers measured at day 0 and 7 post immunisation. In the M. butyricum immunised group, Line ND3-L showed significantly higher specific antibody response to M. butyricum, and this result agrees well with the hypothesis that the Th-1 pathway was expected to be selected for in this line. CONCLUSION: This study has shown that the two different antigens KLH and M. butyricum gave rise to different responses in the set of selected lines, and that the response was only enhanced for the antigen associated with the same response mechanism as that for the trait (ND3, PHA or CC) for which the line was selected. Interactions between innate and acquired immunity have been observed mainly for the high antibody selected trait, indicating there was a specific interaction due to the selection criterion. Furthermore, the results confirmed the independence between the three selected traits. Finally, principal component analysis contributed to visually discriminate high and low responders to the two new antigens in the four lines.

Genetic aspects of auto-immune profiles of healthy chickens.

Auto-antibody profiles binding liver antigens differed between chicken lines divergently selected for specific antibody responses to SRBC, and were affected by ageing suggesting both genetic and environmental effects. Presence and levels of IgM and IgG antibodies binding chicken liver cell lysate (CLL) fragments in plasma at 5 weeks of age from 10 individual full sibs and their parents from 5 Hsrbc and 5 Lsrbc line families was studied to reveal genetic relations. Non-genetic maternal effects were studied by comparing auto-antibody profiles of 36 weeks old hens from 2 other unrelated lines with the profiles from their chicks at hatch. IgM and IgG antibodies from parents and progeny from both Hsrbc and Lsrbc lines bound CLL fragments. Significant line and generation differences and their interactions were found for both isotypes. Higher staining of CLL fragments was usually found for Hsrbc line birds. Lines were clustered by auto-antibody profiles, but staining by birds of both lines in both generations was very individual for IgG and IgM. The current data with full sibs therefore not supported a genetic basis for auto-antibody profiles. IgG but not IgM auto-antibody profiles of chicks correlated with maternal auto-antibody profiles. The results suggest that the auto-antibody repertoire of healthy chickens is largely stochastically initiated and may be affected by environmental challenges during ageing, but genetic mechanisms may underlie staining intensity of individual bound CLL fragments. The present results suggest that identification of fragments or profiles to be used at early age for genetic selection for health traits is not feasible yet. Secondly, the IgM profile of neonatal chickens seems non-organised independent of the maternal profile, but the neonatal IgG profile is much more related with the maternal profile. Consequences of these findings for disease susceptibility or breeding for optimal health are discussed.

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.

Cold stress equally enhances in vivo pro-inflammatory cytokine gene expression in chicken lines divergently selected for antibody responses.

The effects of cold stress, immunization and genetic selection on the expression of mRNA for cytokine genes in poultry have not been completely elucidated. Therefore, in the present experiment, using real-time quantitative RT-PCR, we evaluated the effect of cold stress and immunization with complete Freund's adjuvant (CFA) on expression of mRNA for pro-inflammatory (interleukin-1beta [IL-1beta], IL-6, IL-12beta), Th(1) (IFN-gamma and IL-2), and Th(2) (IL-4 and IL-10) cytokine genes in peripheral blood leukocytes (PBL) of chicken lines divergently selected for either high or low antibody responses. Irrespective of the duration, cold stress enhanced expression of mRNA for IL-1beta, IL-6, IL-12beta and IL-4 cytokine genes in both selection lines. These results indicate that cold stress stimulates both the innate and parts of the adaptive cellular immune system. Immunization with CFA resulted in higher expression of mRNA for pro-inflammatory cytokines and lower expression of mRNA for both Th(1) and Th(2) cytokines.

Display and selection of chicken IgA Fab fragments.

Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicken IgA, using phage display, is described. A novel phagemid vector (pChick3) for the display and selection of chicken IgA antibodies in Fab format was developed. The functionality of pChick3 was demonstrated by construction of an immune antibody library using B cells from chickens infected with Eimeria acervulina. From this library, 10 different IgA fragments with specific binding to the E. acervulina antigen mix, the sporozoite or oocyst fractions were selected. These results demonstrate the efficiency and versatility of the pChick3 vector system that can readily be applied to construct libraries and subsequently select antibodies of the alpha isotype against a wide variety of pathogens and parasites.

A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity.

Animals are continuously threatened by pathogens entering the body through natural openings. Here we show that in chicken ( Gallus gallus ), secretory IgA (sIgA) protects the epithelia lining these natural cavities. A gene encoding a chicken polymeric Ig receptor ( GG-pIgR ), a key component of sIgA, was identified, and shown to be expressed in the liver, intestine and bursa of Fabricius. All motifs involved in pIgR function are present, with a highly conserved Ig-binding motif in the first Ig-like domain. Physical association of GG-pIgR with pIgA in bile and intestine demonstrates that this protein is a functional receptor. Thus, as shown for mammals, this receptor interacts with J-chain-containing polymeric IgA (pIgA) at the basolateral epithelial cell surface resulting in transcytosis and subsequent cleavage of the pIgR, releasing sIgA in the mucosal lumen. Interestingly, the extracellular portion of GG-pIgR protein comprises only four Ig-like domains, in contrast with the five domain structure found in mammalian pIgR genes. The second Ig-like domain of mammalian pIgR does not have an orthologous domain in the chicken gene. The presence of pIgR in chicken suggests that this gene has evolved before the divergence of birds and reptiles, indicating that secretory Igs may have a prominent role in first line defence in various non-mammalian species.