Genetic parameters of IgM and IgG antibodies binding autoantigens in healthy chickens.

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.

Effect of maternal dry period length on colostrum immunoglobulin content and on natural and specific antibody titers in calves.

The objective was to study the effect of dry period length in dairy cows on immunoglobulin content and natural antibodies (NAb) titers in colostrum, growth, and plasma natural and specific antibody titers in plasma of calves. Holstein-Friesian dairy cows (n=167) were randomly assigned to 3 dry period lengths (0, 30, or 60 d). Colostrum production, concentration of colostrum IgG and IgM, and titers of NAb (isotypes IgG and IgM) binding keyhole limpet hemocyanin (KLH) and human serum albumin (HuSA) in colostrum were measured. Female calves were immunized with both KLH and HuSA at wk 6 and 10 of life. Titers of NAb and specific antibody (SpAb) for isotypes IgG, IgM, and total immunoglobulin (IgT) binding KLH or HuSA were determined in plasma of female calves. Primary and secondary antibody responses to KLH or HuSA from wk 6 and 10 were expressed as the increase in antibody titers to wk 10 and 11 of life after primary and secondary challenges, respectively. Pregnancy length for cows with a 0-d dry period was 3d shorter compared with cows with a 30- or 60-d dry period. Birth weight of calves from cows with a 0-d dry period was lower compared with calves from cows with a 30-d dry period. Growth of calves until 12 wk of life was not affected by dry period length. Colostrum production and IgG and IgM concentration in colostrum were lower for cows with a 0-d dry period than a 60-d dry period. Natural IgG and IgM titers binding KLH or HuSA were lower in colostrum from cows with a 0-d dry period compared with cows with a 60-d dry period. Natural antibody titers (IgG, IgM, and IgT) binding KLH or HuSA in plasma were lower during the first 2 wk of life for calves from cows with a 0-d dry period compared with calves from cows with a 30- or 60-d dry period. After primary and secondary immunization of calves with KLH and HuSA, SpAb titers of calves were not affected by dry period length. After secondary immunization, the response of IgG and IgT binding KLH was higher in plasma of calves from cows with a 0-d dry period. The results of this study demonstrate that, although omission of the dry period of dairy cows leads to lower plasma NAb titers in calves during the first 2 wk of life, SpAb titers in calves were not affected and even the secondary antibody responses were enhanced compared with calves from cows with a 30- or 60-d dry period.

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

Environmental particles enter the chicken via several routes. Entry via the respiratory and cloacal routes likely activates immune responses. We studied the localization of simultaneous intratracheally and cloacally applied beads of 2 sizes in the chicken body in time, and when possible, semiquantified the amount of beads. Ten broiler hens, 3.5 wk of age, received 1.25 × 10(9) 1.0-µm beads and 1.05 × 10(7) 10-µm fluorescein isothiocyanate (green) labeled cloacally, and simultaneously the same number and same sizes of tetramethylrhodamine isothiocyanate (red) labeled beads intratracheally. The bursa of Fabricius, lung, liver, kidney, gallbladder, spleen, thymus, small intestine (upper ileum), cecum, intestinal luminal contents, aerated bones, feces, and blood, from 2 chickens per moment were sampled at 1 h, 6 h, 24 h, 48 h, and 1 wk after challenge and studied for the presence of beads using fluorescence microscopy. The highest amount of beads was found in organs closest to the application site after 1 h (i.e., the lungs for red beads, and the bursa for green beads). All tissue samples showed all 4 types of beads at all time moments, most of them within 1 h. Lower levels of beads were found in lungs and bursa after 6 h and in all other organs after 24 h, except for the kidneys where levels declined after 48 h. Surprisingly, beads were found in thymus tissue and only relatively few beads were found in the spleen. At 1 h, 1-µm intratracheally applied red beads were also found in the cecal luminal content and cecal tissue, but not in the small intestinal luminal content, suggesting that ceca are capable of excreting small particles entering the body via the respiratory route. The presence of nondegradable and nonimmunogenic beads of different sizes in all sampled organs throughout the whole chicken body for 7 d suggested potentially negative chronic health and welfare risks for the chicken of environmental particles.

Effects of 2 size classes of intratracheally administered airborne dust particles on primary and secondary specific antibody responses and body weight gain of broilers: a pilot study on the effects of naturally occurring dust.

We studied the effects of a concurrent challenge on slow-growing broilers with 1) airborne particles of 2 sizes: fine dust (smaller than 2.5 microns) and coarse dust (between 2.5 and 10 microns) that were directly collected from a broiler house and 2) lipopolysaccharide on intratracheal immunizations with the specific antigen human serum albumin (HuSA) and measured primary and secondary systemic (total) antibody responses and (isotype-specific) IgM, IgG, and IgA responses at 3 and 7 wk of age. All treatments affected immune responses at several ages, heart morphology, and BW gain, albeit the latter only temporarily. Dust particles significantly decreased primary antibody (IgT and IgG) responses to HuSA at 3 wk of age but enhanced IgM responses to HuSA at 7 wk of age. Dust particles decreased secondary antibody responses to HuSA, albeit not significantly. All of the birds that were challenged with dust particles showed decreased BW gain after the primary but not after the secondary challenge. Relative heart weight was significantly decreased in birds challenged with coarse dust, fine dust, lipopolysaccharide, and HuSA at 3 wk of age, but not in birds challenged at 7 wk of age. Morphology (weight, width, and length) of hearts were also affected by the dust challenge at 3 wk of age. The present results indicate that airborne dust particles obtained from a broiler house when intratracheally administered at an early age affect specific humoral immune responsiveness and BW gain of broilers to simultaneously administered antigens differently than when administered at a later age. The hygienic status of broiler houses at a young age may be of importance for growth and immune responsiveness, and consequently, for vaccine efficacy and disease resistance in broilers. The consequences of our findings are discussed.

Effects of repeated intratracheally administered lipopolysaccharide on primary and secondary specific antibody responses and on body weight gain of broilers.

Earlier, we reported that pathogen-associated molecular patterns such as lipopolysaccharide (LPS), when administered intratracheally (i.t.), affected primary and secondary specific antibody responses to antigens administered concurrently, either i.t. or systemically, and also affected BW gain (BWG) of layers and broilers. In the present study, we evaluated the effects of repeated i.t. challenge with LPS concurrently with or before i.t. immunizations with the specific antigens human serum albumin (HuSA) and rabbit gamma globulin (RGG) on primary (HuSA, RGG) and secondary (HuSA) systemic antibody responses and (isotype) IgM and IgG responses at 2 different ages. Broilers were challenged via the trachea at 3 and 7 wk of age with various combinations of LPS, HuSA, and RGG. All treatments affected immune responses at several time points and also affected BWG, albeit temporarily for the latter. Lipopolysaccharide enhanced primary antibody responses to HuSA and to RGG, when challenged concurrently, but birds challenged solely with LPS at 3 wk of age also showed enhanced primary antibody responses to HuSA and RGG given at 7 wk of age. This was true for IgM as well as IgG isotype responses. Lipopolysaccharide challenge negatively affected BWG at 3 wk of age, whereas the negative effects of LPS after a secondary LPS challenge at 7 wk of age were most pronounced in the birds challenged with LPS at 3 wk of age. The present results indicated that LPS, when administered i.t. at a young age, may affect specific humoral immune responsiveness to antigens administered simultaneously and to BWG of broilers, but also when challenged 4 wk later with specific antigens, suggesting an enhanced status of immune reactivity or sensitivity. The hygienic status of broiler houses at a young age may thus influence BWG, immune responsiveness, and, consequently, the vaccine efficacy and disease resistance in broilers at later ages. The consequences of our findings are discussed.

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

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

Effect of early life thermal conditioning and immune challenge on thermotolerance and humoral immune competence in adult laying hens.

Effects of early life experience with climatic (heat) and hygienic [lipopolysaccharide (LPS)] stress on adaptability to the same stressors in later life were studied in laying hens. Chicks were exposed to 37 degrees C for 24 h at d 5 of age (n = 12) or were i.v.-administered once with 1 mg/kg of BW of LPS at 6 wk of age (n = 12), whereas a control group was reared under standard conditions receiving a placebo treatment of PBS (n = 36). At 24 wk of age, hens treated in early life were reexposed to the same stressor. Early life control hens were exposed to heat stress (n = 12), i.v.-administered with LPS (n = 12), or not exposed (n = 12). To evaluate improvement of adaptability, effects of climatic and hygienic stress on performance, humoral immune competence, and endocrine responsiveness were investigated in hens with early life experience to the stressors and hens only exposed to the stressors in later life. Early life heat exposure did not affect performance, immune, and endocrine parameters. Treatment x time interactions were found for level of antibody (Ab) binding to LPS and keyhole limpet hemocyanin (KLH) after LPS administration, indicating that hens with early life LPS experience differed in response level (Ab binding to LPS) and response pattern (Ab binding to LPS and KLH) compared with hens administered with LPS only at adult age. Our data suggest that early life heat stress exposure did not affect adaptability of laying hens to heat stress in later life. However, early life LPS exposure affected kinetics and magnitude of Ab levels binding to LPS and KLH, indicating that early life LPS exposure can enhance the status of immune reactivity or induce a higher sensitivity to LPS.

Genetic parameters for levels of natural antibodies in chicken lines divergently selected for specific antibody response.

The present study estimated the heritability of natural antibody (NAb) levels binding rabbit red blood cells at the day of immunization with SRBC (NAb0) and 5 d postimmunization (NAb5) in 2 chicken lines divergently selected for specific antibody levels (SpAb) against SRBC and a randombred control line. In addition, genetic correlations between the levels of NAb binding rabbit red blood cells and levels of SpAb binding SRBC were estimated. The heritability of the SpAb level against SRBC was estimated based on data from 23 generations of selection and in total 21,842 chickens. The heritability of NAb levels against rabbit red blood cells was estimated on 1,764 chickens in generations 22 and 23 using a bivariate analysis including the trait under selection (i.e., SpAb responses to SRBC). Maternal environmental effects were accounted for in the analyses. The heritability for SpAb was 0.17, the heritability for NAb0 levels was 0.23, and the heritability for NAb5 was 0.09. The genetic correlation between SpAb and NAb0 was 0.15 and the genetic correlation between SpAb and NAb5 was 0.50. The weak, though positive, genetic correlation between SpAb responses and the NAb0 levels in nonimmunized chickens, as well as the moderate genetic correlation between SpAb responses and the NAb levels 5 d postimmunization with SRBC, suggest that selection for enhanced adaptive immune system results in a positive correlated response of the innate immunity of chickens.

Effects of dust and airborne dust components on antibody responses, body weight gain, and heart morphology of broilers.

Pathogen-associated molecular patterns (PAMP) such as lipopolysaccharide (LPS), lipoteichoic acid, beta-glucans (BGL), and possibly many others are important parts of (fine) dust in animal houses. When intratracheally (i.t.) administered, PAMP affected specific primary and secondary humoral immune responses to concurrently i.t. or systemically administered antigens and BW gain (BWG) of layer chickens. In the present study, we evaluated the effects of i.t. challenge with various PAMP known to be present in dust: LPS, lipoteichoic acid, zymosan-A (containing 1,3 BGL), next to heat-inactivated dust particles as a representative of mechanical stress, a combination of the former components, and NH3 as a chemical component of dust on primary and secondary (total) systemic antibody (Ab) responses and (isotype) IgM and IgG responses to concurrently i.t.-administered human serum albumin (HuSA) in broilers. Birds were challenged via the trachea for 2 consecutive days at 3 and 7 wk of age, respectively. All treatments affected immune responses at several moments, BWG, and heart morphology. beta-Glucans and LPS affected the birds most pronounced and for a prolonged period. Intratracheally administered LPS and BGL significantly enhanced primary and secondary total Ab, IgM Ab, and IgG Ab responses to HuSA. All birds that were challenged with dust, PAMP, or NH3 concurrently with HuSA showed a decreased BWG especially after primary, but also after secondary challenge. Weight, width, and length of hearts were enhanced in dust and PAMP-treated birds as well when these birds were challenged with HuSA. The present results indicated that components of dust such as PAMP when i.t. administered affect humoral immune responsiveness of broilers, which may lead to an enhanced status of immune reactivity. Furthermore, our results suggest that the hygienic status of the environment influences BWG and may affect heart morphology, and as a consequence physiology in broilers. The consequences of our findings with respect to dust, (airborne) PAMP, hygienic conditions in the barn, and immune responsiveness of broilers are discussed.

Chicken lines divergently selected for antibody responses to sheep red blood cells show line-specific differences in sensitivity to immunomodulation by diet. Part I: Humoral parameters.

Individual differences in nutrient sensitivity have been suggested to be related with differences in stress sensitivity. Here we used layer hens divergently selected for high and low specific antibody responses to SRBC (i.e., low line hens and high line hens), reflecting a genetically based differential immune competence. The parental line of these hens was randomly bred as the control line and was used as well. Recently, we showed that these selection lines differ in their stress reactivity; the low line birds show a higher hypothalamic-pituitary-adrenal (HPA) axis reactivity. To examine maternal effects and neonatal nutritional exposure on nutrient sensitivity, we studied 2 subsequent generations. This also created the opportunity to examine egg production in these birds. The 3 lines were fed 2 different nutritionally complete layer feeds for a period of 22 wk in the first generation. The second generation was fed from hatch with the experimental diets. At several time intervals, parameters reflecting humoral immunity were determined such as specific antibody to Newcastle disease and infectious bursal disease vaccines; levels of natural antibodies binding lipopolysaccharide, lipoteichoic acid, and keyhole limpet hemocyanin; and classical and alternative complement activity. The most pronounced dietary-induced effects were found in the low line birds of the first generation: specific antibody titers to Newcastle disease vaccine were significantly elevated by 1 of the 2 diets. In the second generation, significant differences were found in lipoteichoic acid natural antibodies of the control and low line hens. At the end of the observation period of egg parameters, a significant difference in egg weight was found in birds of the high line. Our results suggest that nutritional differences have immunomodulatory effects on innate and adaptive humoral immune parameters in birds with high HPA axis reactivity and affect egg production in birds with low HPA axis reactivity.

Effect of single or combined climatic and hygienic stress on natural and specific humoral immune competence in four layer lines.

Effects of long-term climatic stress (heat exposure), short-term hygienic stress [lipopolysaccharide (LPS)], or a combination of both challenges on the immune competence of 4 layer lines was investigated. The lines were earlier characterized for natural humoral immune competence and survival rate. Eighty hens per line were randomly divided over 2 identical climate chambers and exposed to a constant high temperature (32 degrees C) or a control temperature (21 degrees C) for 23 d. Half of the hens housed in each chamber were i.v. injected with LPS at d 1 after the start of the heat stress period. Within each of the treatment groups, half of the hens were s.c. immunized with human serum albumin (HuSA) at d 2 after the start of the heat stress period to measure specific antibody (Ab) titers to HuSA. The effect of heat, LPS, or a combined challenge on specific Ab titers to HuSA, natural Ab titers to keyhole limpet hemocyanin or HuSA (in hens that were not immunized with HuSA), and activity of the classical and alternative complement pathways were investigated. Heat stress enhanced specific and natural immune responses. Administration of LPS enhanced natural immune responses but decreased specific immune responses. The lack of interaction between heat stress and LPS administration, except for natural Ab titers to HuSA, suggest that these were 2 independent stressors. The lines had a similar response pattern but differed in the response level. Neither natural humoral immune competence nor survival rate, for which the lines had been characterized, was indicative of the specific and natural immune response to different stressors. Lipopolysaccharide and heat stress initiated sequential responses over time, with an earlier effect of short-term LPS exposure (within the first and second week) and a later effect of long-term heat exposure (within the second and third week). These data suggest that LPS and heat stress affect the natural and specific immune competence of laying hens.

Intratracheally administered pathogen-associated molecular patterns affect antibody responses of poultry.

Various potential immune-modulating microbially derived pathogen-associated molecular patterns (PAMP), or so called homotopes, are present in high concentrations in the environment of food animals. In previous studies, intravenously administered PAMP had variable effects on specific primary and secondary immune responses of poultry to systemically administered antigens. In the present study, we evaluated the effects of intratracheal (i.t.) challenge with the PAMP lipopolysaccharide, lipoteichoic acid (LTA), and Zymosan-A (containing 1,3 beta-glucan) on primary and secondary (total) antibody (Ab) responses and (isotype) IgM, IgG, and IgA responses to systemically administered human serum albumin (HuSA), and Ab titers to infectious bursal disease (Gumboro virus) and infectious bronchitis vaccines in layer hens at 9 and 22 wk of age. Birds were challenged via the trachea with PAMP for 5 consecutive days prior to primary and secondary immunization with HuSA. Intratracheally administered LTA and, to a minor extent, lipopolysaccharide significantly enhanced secondary total and IgG Ab responses to HuSA. 1,3 beta-Glucan did not significantly affect Ab responses to HuSA. All birds challenged with PAMP showed a decreased BW. Higher total Ab titers to infectious bursal disease and infectious bronchitis were found in birds challenged with LTA. The present results indicate that i.t. administered PAMP affect the humoral immune responsiveness of poultry, which may lead to an enhanced status of immune reactivity. Furthermore, our results suggest that the hygienic status of the environment influences BW (gain). The consequences of immune modulation by airborne PAMP or hygienic conditions in chicken husbandry for vaccine delivery and immune responsiveness of poultry are discussed.

Antibody responses to keyhole limpet hemocyanin, lipopolysaccharide, and Newcastle Disease virus vaccine in F2 and backcrosses of white Leghorn lines selected for two different immune response traits.

Planned crosses were designed to produce an F(2) and 2 backcross populations from 2 lines of White Leghorn chickens previously selected over 10 generations for 2 different in vivo immune responses. The selection criteria applied on the 2 grandparental lines were as follows: high antibody response to Newcastle disease virus vaccine 3 wk after vaccination (ND3) and high cell-mediated immune response [response to phytohemagglutinin]. Furthermore a control line was kept by random breeding. The objective of the study was to estimate if the 2 selection criteria applied on the pure lines had changed the level of and type of immune (humoral) response to a new antigen, keyhole limpet hemocyanin (KLH), in the various second-generation progeny groups. In addition, correlations between parameters of acquired and innate immunity were tested. Primary total (IgT) and isotype-specific (IgG and IgM) antibody response to KLH 1 wk after immunization and levels of natural antibodies (NAB) binding to Salmonella enteriditis-derived lipopolysaccharide (LPS) were measured. Although no differences were present between IgM and IgG antibodies to KLH and the phytohemagglutinin skin-swelling response, significant differences were present between all the progeny groups for IgT to KLH and ND3 and NAB binding to LPS. The mean values for IgT to ND3 and KLH were significantly different between the crosses using the selected lines compared with the control line, indicating a contribution of the previous selection. In addition, a sex effect was found for IgM to KLH and NAB to LPS, for which females had a higher response than males in both cases. No interaction between progeny type and sex was found. Furthermore, significant positive correlations were found between NAB to LPS and specific antibody titers to KLH. Finally, the results of the present study demonstrated an interaction between innate and acquired immunity under this strategy of selection and crossbreeding and confirmed the effect of selection on general immune response to a new antigen in second-generation crosses.

Natural humoral immune competence and survival in layers.

The relation between survival and levels of humoral components of innate (and specific) immune competence of laying hens was investigated in a population of 1,063 laying hens from 12 purebred layer lines. Natural immune competence of the chickens was studied by measuring levels of natural antibodies (NAb) binding to keyhole limpet hemocyanin (KLH) or lipopolysaccharide (LPS), respectively, and hemolytic (classical and alternative) complement activity at 20, 40, and 65 wk of age. In addition, levels of antibodies binding a Newcastle disease vaccine strain as a measure of specific immunity were investigated at 20 wk of age. A distinction could be made between lines showing high or low immune competence with respect to NAb, complement activity, and specific antibodies. Within lines, significant correlations were found for each of the innate parameters among the 3 ages. The innate and specific parameters were, however, not correlated with each other. Based on the limited data set, it was not possible to draw conclusions on line differences for innate or specific immune competence in relation to survival. However, regardless of line, low levels of NAb binding to KLH or high levels of NAb binding to LPS were detected in chickens that did not survive the laying period. The major difference between the responses of NAb binding to KLH or LPS was that the chickens probably did not encounter KLH, which suggests a reflection of the capacity to respond, whereas the chickens most probably did encounter LPS, which suggests a reflection of the active status of the innate humoral immune system. In conclusion, we propose that levels (KLH) and activation (LPS) of components of natural antibodies are indicative for the probability that chickens survive a laying period.

Age- and breed-dependent adapted immune responsiveness of poultry to intratracheal-administered, pathogen-associated molecular patterns.

Immune modulation of poultry by airborne pathogen-associated molecular patterns (PAMP) was studied. White and Brown layer chicks were exposed intratracheally during 5 consecutive days at 7 wk of age with Escherichia coli-derived lipopolysaccharide (LPS), Saccharomyces cerevisiae-derived 1,3 beta-glucan (BGL), a combination of both, or PBS as a control. Six weeks later, birds received similar or crossover PAMP treatments. Body weight (gain), feed conversion, (primary and secondary) specific antibody responses to model antigens, and natural antibody levels were measured. In general, BGL enhanced but LPS exposure decreased primary immune responses at 7 wk of age, whereas both PAMP-enhanced secondary immune responses but decreased primary immune responses at 13 wk of age. Body weight gain and feed conversion at both ages were negatively affected by LPS, especially in White birds, but not by BGL. Pathogen-associated molecular patterns exposure at 7 wk of age also affected Ab responses at 13 wk of age. Birds exposed to a combination of LPS + BGL at 7 wk of age had significantly lower secondary total and IgG Ab responses at 13 wk of age. Birds from both breeds showed enhanced BW gain after exposure to LPS at 13 wk of age, when initially challenged at 7 wk of age with LPS, BGL, or a combined challenge with both. Pathogen-associated molecular patterns exposure at 7 wk of age affected humoral immunity and BW gain at 13 wk of age in a positive (BGL) or negative (LPS) fashion. Repeated exposure to PAMP did not affect Ab responses, but crossover exposure to PAMP in general enhanced Ab responses. Body weight gain was positively affected by repeated exposure but not by crossover exposure, suggesting adaptation of the birds to early PAMP exposure. Our findings suggest that sensitivity of poultry for immune modulation by airborne PAMP differs between ages, is breed-dependent, and is not irreversible of nature. In addition, our data suggest different adaptation to hygienic conditions, both with respect to immune reactivity and BW gain.

Severe feed restriction enhances innate immunity but suppresses cellular immunity in chicken lines divergently selected for antibody responses.

The main aim of this study was to investigate the effects of different levels of feed restriction (FR) on immune responses of chicken lines divergently selected for high (H) and low (L) antibody responses to SRBC. We hypothesized that severe feed restriction suppresses immune responses and the level of immune suppression differs between birds with different genetic background. Therefore, we tested antibody responses, blood lymphocyte proliferative responses, and production of reactive oxygen intermediates (ROI) by Zymosan A-stimulated blood cells in chicken lines maintained on 3 levels of FR. The H line birds had significantly higher antibody responses, higher ROI production, and lower corticosterone (CORT) levels when compared with the L line birds. Feed restriction induced no significant effect on specific antibody responses to either a T helper 1- (Mycobacterium butyricum) or a T helper 2- (keyhole limpet hemocyanin) type antigen. Feed-restricted birds showed a marked reduction in natural antibodies binding lipoteichoic acid, in vitro lymphocyte proliferation in response to stimulation with concanavalin A, BW gain, and relative lymphoid organ weights compared with the birds fed ad libitum. However, FR birds showed a markedly enhanced ROI production, and plasma CORT levels compared with the birds fed ad libitum. The enhanced ROI production and suppressed lymphocyte proliferation coinciding with enhanced plasma CORT levels suggest stress-mediated immunomodulating effects of FR. A significant treatment by line interaction was found for ROI production; the increase of ROI production was larger in the H line than in the L line under severe FR. The increase in CORT levels was larger in the L line than in the H line under severe FR. Furthermore, the L line gained more BW than the H line under ad libitum conditions. Finally, under severe FR, relative spleen weight was lower in the L line than in the H line. The present findings suggest genetic differences affecting physiological and immunological responses under FR conditions.

Homotopes affect primary and secondary antibody responses in poultry.

Activation of the innate immune system by different homotopes may direct the subsequent specific immune response, and as a consequence may have significant implications on vaccination. In this study, effects of i.v. administered lipopolysaccharide (LPS), and lipoteichoic acid (LTA), respectively, and s.c. administered Keyhole Limpet Hemocyanin (KLH) on total and isotype-specific (IgM and IgG) primary and secondary antibody responses of chickens to human serum albumin (HuSA) were determined. Similarly, effects of pre-treatment of the birds with the homotopes on primary antibody responses to rabbit gamma globulin (RGG) were measured. In addition, antigen-specific (HuSA and RGG) and mitogenic (concanavalin A, Con A) responses of peripheral blood leucocytes in vitro were also measured. Our findings confirm earlier observations that both LTA as well as LPS have immunomodulatory features in chickens, albeit in an opposite direction. LTA enhanced primary and to a minor degree secondary antigen-specific antibody titers, but LPS was found to suppress these responses. In RGG-primed birds, LPS enhanced a non-antigen-specific recall response to HuSA. In addition, KLH acted in an immunomodulatory role too, enhancing primary and secondary antibody responses, but suppressing non-specific cellular immunity in a non-antigen-specific fashion. Possible mechanisms underlying our observations, and the role of LTA, LPS, and KLH in polarization of the specific antibody response in chickens are discussed.

Serological screening for MHC (B)-polymorphism in indigenous chickens.

As part of a series of studies to characterize innate and specific immune responses of indigenous chicken lines, birds from Bolivia and India were screened serologically for MHC class IV (BG) polymorphism by direct haemagglutination using haplotype-specific antisera (B2, B4, B12, B13, B14, B15, B19, B21). The sample consisted of 95 Bolivian indigenous chickens and 119 hens from the four most common North Indian 'back-yard' chicken lines: Yellow Aseel (AP), Kadaknath (KN), frizzled typed (Ff-) and naked neck (NN). Of all chickens tested, the majority were haplotyped as B2, B15, B19 and B21. Of the Bolivian chickens, 89.5% could be haplotyped: 54.9% were homozygous (including 43.3% B15), and 34.6% were heterozygous (including 15.7% B15). B2-like haplotypes were not found among the Bolivian hens, and only 3.2% of these birds showed homozygous B21-like proteins. Of the Indian hens, MHC (BG)-like proteins could be detected in 60.0% of the AP birds, 6.7% of the KN birds; 40.0% of the Ff- birds; and 10.3% of the NN birds. In these lines, a total of 40.1% (AP), 6.7% (KN), 30.1% (Ff-) and 10.3% (NN) were homozygous for the B-haplotype. Only in the AP line (19.9%), and the Ff- line (9.9%) were heterozygous B-haplotypes types found. The B2 haplotype was found in all Indian chicken lines. Most Indian birds have completely unknown haplotypes, indicating a potentially interesting genetic pool. Subgrouping the Bolivian and Indian indigenous hens into monomorphic BG populations revealed individual differences based on the B-types.

Haemolytic complement activity and humoral immune responses to sheep red blood cells in indigenous chickens and in eight German Dahlem Red chicken lines with different combinations of major genes (dwarf, naked neck and frizzled) of tropical interest.

A total of 376 chickens from different ecotypes were immunized with the non-pathogenic multi-determinant antigen sheep red blood cells (SRBC). The ecotypes included indigenous chickens from various locations in Tanzania (n=102), India (n=86) and Bolivia (n=89). In addition, eight German Dahlem Red (GDR) chicken lines with different major genes (dwarf, naked neck and frizzled) of tropical interest were also immunized with SRBC. Immune competence of the breeds was assessed by measuring complement haemolytic activity, both from the classical calcium-dependent complement pathway (CPW) and alternative calcium-independent complement pathway (APW), alongside IgTotal, IgG and IgM antibody responses to SRBC at 7 days post immunization. Large variations in complement activity and antibody responses to SRBC were observed within and between the indigenous breeds. Many indigenous chickens, especially from Bolivia, showed decreased complement activity (APW) following immunization with SRBC. Breeds from India showed the highest CPW activity and humoral (especially IgM) responses to SRBC, suggesting high immune competence. In contrast, Bolivian chickens were characterized by low CPW activity, low APW activity and low antibody levels to SRBC suggesting an overall low immune competence. In the GDR chickens, characterized by high CPW activity and high IgG antibody responses to SRBC, the major genes for naked neck, frizzling and dwarfism had no significant effect on the antibody responses and complement activity to SRBC.

Serum haemolytic complement levels in German Dahlem Red chickens are affected by three major genes (naked neck, dwarf, frizzled) of tropical interest.

German Dahlem Red chickens with three different major genes of tropical interest: Nana- (naked neck), Ff- (frizzled) and dw- (dwarf), respectively, were tested for serum haemolytic complement, which is essential in innate host defence against infectious agents. Eight different combinations of genes for body size and feather coverage were evaluated. Significant differences both for both the calcium-dependent (classical, CPW) and the calcium-independent (alternative, APW) complement titres were found between the phenotypes. Phenotype nanaffDw- showed the highest complement status. The frizzled (Ff-) gene had a negative influence on APW titres, whereas the dwarf (dw-) gene had a negative influence on CPW titres. The naked neck (Nana-) gene had various influences on the haemolytic complement status. All tested hens had MHC (B) 21 haplotypes, whereas the gene for dwarfism appeared to be linked with the B19 haplotype. It was concluded that introducing major genes (Nana-, dw-, Ff-) to conquer environmental stress in hot climates can have a negative impact on certain aspects of the innate immunity of poultry.