Lactobacilli-Based Pro and Postbiotic Efficacy Are Also Influenced by Other Factors Than Dietary Challenging Conditions.

The present study aimed to confirm the previously reported 'recovery' effect to a challenging diet (CD) of a Lactobacilli-based probiotic (Pro) and its derived postbiotic (Post) in broilers. Identical diet compositions were used, and observations were extended to a second CD diet. A completely randomised block design of 2 × 3 treatment groups with two CDs and three additive conditions (Control, Pro, Post) was used. One additional group received a standard diet (SD). The study involved 1600 one-day-old Ross 308 male broilers. All diets, fed from d1 to 35, were formulated to contain identical nutrients levels, with CDs formulated to be greater than SD in nonstarch polysaccharides using rye and barley (Rye CD) or dry distiller grains with soluble, sunflower and rapeseed meal (DDGS CD). Growth performance parameters, footpad lesions (FPL) score and plasma Ca, P and uric acid concentrations were measured. Compared to SD, birds fed Rye CD and DDGS CD had a higher 1-35 days feed conversion ratio (+3.4 and +4.1%, respectively), due to a higher feed intake for Rye CD (+2.9%) and a lower body weight for DDGS CD (-4.1%). An effect of additive was restricted to Rye CD where Post depressed BW at d28 and d35 (-3.7 and -2.4%, respectively). Compared to Rye CD, DDGS CD lowered plasma Ca/P at d21 (-9.0%) and d35 (-8.1%) and uric acid at d21 (-26%). Pro increased plasma Ca in Rye CD at d21 (+12%) and Post decreased plasma uric acid in DDGS CD at d35 (-25%). All other plasma parameters were not affected. The previously observed recovery effect of a commercial probiotic and postbiotic were not reproducible under highly similar growth conditions, which suggests that both may have specific physiological effects which are only expressed under specific circumstances.

Maternal Fish Oil Supplementation Affects the Social Behavior, Brain Fatty Acid Profile, and Sickness Response of Piglets.

BACKGROUND: Maternal dietary docosahexaenoic acid (DHA) intake is thought to affect development in the offspring. OBJECTIVE: We assessed the impact of maternal dietary DHA on behavior, brain fatty acid (FA) profile, and sickness response of offspring in pigs, a pertinent model for human nutrition. METHODS: Sows (n = 24) were fed a diet with DHA-rich fish oil (FO) (20 g/kg) or high-oleic acid sunflower oil (HOSF) (20 g/kg) from day 61 of gestation through lactation. At 4 wk of age, 4 piglets per litter were weaned and mixed with piglets from other litters. Behavior was observed in 4- to 8-wk-old piglets, and brain FA composition was analyzed at 4 (n = 15) and 14 (n = 12) wk. Thirteen-week-old piglets (n = 48) were subjected to a lipopolysaccharide (LPS) challenge. Body temperature, plasma cytokines, and motivation to approach a familiar human, indicative of a sickness response, were measured. RESULTS: FO-fed pigs displayed more social activities (+262%, P = 0.02), played more (+61%, P = 0.03), and tended to show fewer oral manipulative behaviors directed at pen mates (-25%, P = 0.06) than did HOSF-fed pigs up to 4 wk after weaning. Brain DHA concentrations were higher in FO- than in HOSF-fed pigs up to 10 wk after supplementation (+10-50%, P < 0.001), although differences declined with age. Body temperature (P < 0.001) and tumor necrosis factor α and interferon Î³ concentrations (P < 0.05) increased after LPS injection, but no diet effect was found (P > 0.10). LPS-treated pigs were less likely to approach the human than saline-treated pigs in the HOSF-fed (-29%, P = 0.0003), but not in the FO-fed group (-13%, P = 0.11). CONCLUSIONS: Maternal DHA beneficially affected offspring social behavior after weaning and mildly attenuated sickness behavior after an inflammatory challenge in pigs. These behavioral changes may be mediated by increased brain DHA proportions.

Microbiota but not immune modulation by a pro- and postbiotic was associated with the diet-additive interaction in broilers.

This study investigated the diet-additive interactions of a Lactobacilli-based probiotic (Pro) and postbiotic (Post) on immune parameters and cecal microbiota composition, with subsequent effects on the metabolome in broilers. A completely randomized block design was employed with 2 diets [standard (SD), and challenge (CD)] and 3 additive conditions (Control, Pro, Post) involving 1,368 one-day-old male Ross 308 broilers equally distributed among 36 pens in a 42 d study. Diets were formulated to contain identical nutrient levels, with CD higher than SD in non-starch polysaccharide content by including rye and barley. Total non-specific serum Ig A, M and G concentrations were determined weekly from d14 to 35. Following vaccination, titres of specific antibodies binding Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) were measured. Microbiota composition was analyzed by 16S rRNA gene sequencing at d14 and 35, and α- and ß-diversity indexes (Observed, Chao1, Bray, Jaccard) were calculated. Cecal short-chain fatty acids and the semi-polar metabolome were determined in the Control SD and all CD groups at d35. At d35, a diet-additive interaction was observed on cecal microbiota composition. Within SD, Pro and Post did not affect operational taxonomic units (OTU) abundance (adjusted-P > 0.05) and diversity indexes (P > 0.05). Within CD, Pro and Post affected the relative abundances of 37 and 44 OTUs, respectively (adjusted-P < 0.05), with Post but not Pro affecting ß-diversity indexes (P = 0.041 and 0.064 for Bray and Jaccard, respectively). Within CD, Post increased cecal acetate (21%; P = 0.007) and butyrate (41%; P = 0.002) concentration and affected the concentration of 2 metabolites (adjusted-P < 0.05), while Pro affected 240 metabolites (adjusted-P < 0.05). No diet-additive interactions were observed on serum Ig (P > 0.05), except for IgM at d14 (P = 0.004). Diet composition, but not the additives, affected immune status parameters. The Pro and Post affected cecal microbiota composition only under dietary challenging conditions as previously reported for growth.

Diet composition influences probiotic and postbiotic effects on broiler growth and physiology.

Dietary ingredient and nutrient composition may affect the efficacy of additives in broilers. Specific feed ingredients can represent dietary challenging conditions for broilers, resulting in impaired performances and health, which might be alleviated by dietary probiotics and postbiotics. We assessed the effects of a Lactobacilli probiotic (Pro) and postbiotic (Post) when added to a standard (SD) and challenge (CD) diet. A completely randomized block study with 2 diets (SD, CD) and 3 additive conditions (Control, Pro and Post) involving 1,368 one-day-old Ross male broilers, equally distributed among 36 pens, from d1 to d42 was conducted. Both diets were formulated to contain identical levels of nutrients, with CD formulated to be richer than SD in nonstarch polysaccharides using rye and barley as ingredients. Readout parameters included growth performance parameters, footpad lesions score, blood minerals and biochemical parameters, and tibia health, strength, and composition. Compared to SD, CD decreased BW (1,936 vs. 2,033 g; p = 0.001), increased FCR (p < 0.01) and impaired tibia health and strength (p < 0.05) at d35, thereby confirming the challenging effect of CD. Pro and Post increased BW in CD (+4.7 and +3.2%, respectively, at d35; P < 0.05) but not in the SD group, without affecting FCR. Independently of the diet, Pro increased plasma calcium, phosphorus and uric acid at d21 (+6.2, +7.4, and +15.5%, respectively) and d35 (+6.6, +6.2 and +21.0%, respectively) (P < 0.05) while Post increased plasma magnesium only at d21 (+11.3%; P = 0.037). Blood bile acids were affected by additives in an age- and diet-dependent manner, with some opposite effects between dietary conditions. Diet composition modulated Pro and Post effects on broiler growth performance. Additionally, Pro and Post affected animal metabolism and leg health diet-dependently for some but not all investigated parameters. Our findings show that the effects of pro- and postbiotics on the growth performance and physiology of broilers can be dependent on diet composition and thus possibly other factors affecting diet characteristics.

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.

Transgenerational effects of innate immune activation in broiler breeders on growth performance and immune responsiveness.

The impact of transgenerational effects on growth performance and immunity has not yet been studied extensively within the poultry husbandry sector. An important factor is the impact of the hens on the physical well-being and fitness to the environment of the offspring. This study is the first to investigate the effect of stimulating the maternal innate immune system with lipopolysaccharides (LPS) or ß-glucan on growth performance and immune responses in the next generation. Transgenerational effects and consequences of these maternal treatments were further examined using a necrotic enteritis (NE) challenge model in the offspring. We show that offspring of LPS-treated broiler breeders have a higher feed efficiency from 14 to 21 days of age, that is, the period just after the NE challenge. Moreover, more broiler chickens with intestinal lesions after the NE challenge were found in the offspring of the LPS-treated broiler breeders. Both the LPS and ß-glucan maternal treatments resulted in transgenerational effects on blood-derived monocytes by showing a tendency of decreased IL1ß mRNA levels after ex vivo LPS stimulation. These data are a first indication that broiler breeder hens can affect immune responsiveness and feeding efficiency of their offspring in a transgenerational manner.

Low Incubation Temperature During Late Incubation and Early Feeding Affect Broiler Resilience to Necrotic Enteritis in Later Life.

Resilient animals can cope with environmental disturbances in life with minimal loss of function. Resilience can be enhanced by optimizing early-life conditions. In poultry, eggshell temperature (EST) during incubation and early feeding are two early-life conditions that are found to alter neonatal chick quality as well as immune response in later life. However, whether these early-life conditions affect disease resilience of chickens at later ages has never been studied yet. Hence, we studied the effects of EST [(37.8°C (control) or 36.7°C (lower)] during late incubation (≥embryonic days 17-19.5) and feeding strategy after hatch [immediately (early feeding) or 51-54 h delayed (delayed feeding)] on later-life broiler resilience in a 2 × 2 factorial arrangement. At hatch, 960 broilers of both sexes from a 54-week-old Ross breeder flock were equally divided over 32 pens (eight replicate pens per treatment combination) and grown for 6 weeks. Necrotic enteritis was induced by a single inoculation of Eimeria spp. at d 21 and repeated Clostridium perfringens inoculation (3×/d) during d 21-25. Mortality and body weight (BW) gain were measured daily during d 21-35 as indicators of resilience. Additionally, disease morbidity was assessed (gut lesions, dysbacteriosis, shedding of oocysts, footpad dermatitis, and natural antibody levels in blood). Results showed a lack of interaction between EST and feeding strategy for the vast majority of the variables. A lower EST resulted in lower BW gain at d 5 and 8 post Eimeria inoculation (P = 0.02) and more Eimeria maxima oocysts in feces at d 8 post Eimeria inoculation compared to control EST (P < 0.01). Early feeding tended to lower mortality compared to delayed feeding (P = 0.06), but BW gain was not affected by feeding strategy. Morbidity characteristics were hardly affected by EST or feeding strategy. In conclusion, a few indications were found that a lower EST during late incubation as well as delayed feeding after hatch may each impair later-life resilience to necrotic enteritis. However, these findings were not manifested consistently in all parameters that were measured, and conclusions are drawn with some restraint.

Innate immune training and metabolic reprogramming in primary monocytes of broiler and laying hens.

Recently, we have reported trained innate immunity in laying chicken monocytes. In the present study, we further investigated trained innate immunity of monocytes in layers and broilers. Monocytes of both breeds isolated from blood were trained in vitro with ß-glucan, rec-chicken IL-4 or a combination of both, and restimulated with lipopolysaccharide (LPS), after which inflammation and metabolism-related responses were measured. Training of laying and broiler hen monocytes resulted in increased mRNA levels of IL-1ß, iNOS and HIF-1α, but enhanced surface expression of CD40 and NO production was only observed in layers. Our in vitro study demonstrates that monocytes from different genetic backgrounds can be trained. However, the observed differences suggest a differential effect on immune functionality associated with innate training. Whether these differences in immune functions between layers and broilers have effect on disease resistance remains to be elucidated.

Training of Primary Chicken Monocytes Results in Enhanced Pro-Inflammatory Responses.

Beta-glucan-stimulated mammalian myeloid cells, such as macrophages, show an increased responsiveness to secondary stimulation in a nonspecific manner. This phenomenon is known as trained innate immunity and is important to prevent reinfections. Trained innate immunity seems to be an evolutionary conserved phenomenon among plants, invertebrates and mammalian species. Our study aimed to explore the training of primary chicken monocytes. We hypothesized that primary chicken monocytes, similar to their mammalian counterparts, can be trained with ß-glucan resulting in increased responses of these cells to a secondary stimulus. Primary blood monocytes of white leghorn chickens were primary stimulated with ß-glucan microparticulates (M-ßG), lipopolysaccharide (LPS), recombinant chicken interleukin-4 (IL-4) or combinations of these components for 48 h. On day 6, the primary stimulated cells were secondary stimulated with LPS. Nitric oxide (NO) production levels were measured as an indicator of pro-inflammatory activity. In addition, the cells were analyzed by flow cytometry to characterize the population of trained cells and to investigate the expression of surface markers associated with activation. After the secondary LPS stimulation, surface expression of colony stimulating factor 1 receptor (CSF1R) and the activation markers CD40 and major histocompatibility complex class II (MHC-II) was higher on macrophages that were trained with a combination of M-ßG and IL-4 compared to unstimulated cells. This increased expression was paralleled by enhanced NO production. In conclusion, this study showed that trained innate immunity can be induced in primary chicken monocytes with ß-glucan, which is in line with previous experiments in mammalian species. Innate immune training may have the potential to improve health and vaccination strategies within the poultry sector.

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.

Early-life microbiota transplantation affects behavioural responses, serotonin and immune characteristics in chicken lines divergently selected on feather pecking.

Gut microbiota influences host behaviour and physiology, such as anxiety, stress, serotonergic and immune systems. These behavioural and physiological characteristics are related to feather pecking (FP), a damaging behaviour in chickens that reduces animal welfare and productivity. Moreover, high FP (HFP) and low FP (LFP) lines differed in microbiota composition. However, it is unknown whether microbiota can influence the development of FP. For the first time, we identified the effects of microbiota transplantation on FP, and behavioural and physiological characteristics related to FP. HFP and LFP chicks received sterile saline (control), HFP or LFP microbiota transplantation during the first two weeks post-hatch. Microbiota transplantation influenced behavioural responses of the HFP line during treatment and of the LFP line after treatment. In both lines, homologous microbiota transplantation (i.e., receiving microbiota from their line) resulted in more active behavioural responses. Furthermore, microbiota transplantation influenced immune characteristics (natural antibodies) in both lines and peripheral serotonin in the LFP line. However, limited effects on microbiota composition, stress response (corticosterone) and FP were noted. Thus, early-life microbiota transplantation had immediate and long-term effects on behavioural responses and long-term effects on immune characteristics and peripheral serotonin; however, the effects were dependent on host genotype. Since early-life microbiota transplantation influenced behavioural and physiological characteristics that are related to FP, it could thus influence the development of FP later in life.

Stress response, peripheral serotonin and natural antibodies in feather pecking genotypes and phenotypes and their relation with coping style.

Feather pecking (FP), a serious welfare and economic issue in the egg production industry, has been related to coping style. Proactive and reactive coping styles differ in, among others, the stress response, serotonergic activity and immune activity. Yet, it is unknown whether genetic lines divergently selected on FP (i.e. FP genotypes) or individuals differing in FP (i.e. FP phenotypes) can be categorized into coping styles. Therefore, we determined peripheral serotonin (5-HT) levels, natural antibody (NAb) titers, behavioral and corticosterone (CORT) responses to manual restraint (MR) in FP genotypes (high FP (HFP), low FP (LFP) and unselected control (CON) line) and FP phenotypes (feather pecker, feather pecker-victim, victim and neutral). We further examined the consistency of and relationships between behavioral and physiological measures. FP genotypes differed in behavioral responses to MR, 5-HT levels and NAb titers, but not in CORT levels after MR. HFP birds had less active responses at adolescent age, but more active responses at adult age compared to LFP and CON birds. The CON line had higher 5-HT levels at adolescent age, while the HFP line had lower 5-HT levels than the other lines at adult age. Overall, the HFP line had lower IgM NAb titers, while the LFP line had lower IgG NAb titers compared to the other lines. FP phenotypes differed in behavioral responses to MR and 5-HT levels, but not in CORT levels after MR or NAb titers. Within the HFP line, feather peckers tended to have less active responses compared to neutrals at adolescent age, while victims had more active responses compared to the other phenotypes at adult age. Feather peckers had higher 5-HT levels than neutrals at adult age. Behavioral and CORT responses to MR were not consistent over time, suggesting that responses to MR might not reflect coping style in this study. Furthermore, proactive behavioral responses were correlated with reactive physiological measures and vice versa. Thus, it was not possible to categorize FP genotypes or FP phenotypes into specific coping styles.

Differences in gut microbiota composition of laying hen lines divergently selected on feather pecking.

Feather pecking (FP), a damaging behavior where laying hens peck and pull at feathers of conspecifics, is multifactorial and has been linked to numerous behavioral and physiological characteristics. The gut microbiota has been shown to influence host behavior and physiology in many species, and could therefore affect the development of damaging behaviors, such as FP. Yet, it is unknown whether FP genotypes (high FP [HFP] and low FP [LFP] lines) or FP phenotypes (i.e., individuals differing in FP, feather peckers and neutrals) differ in their gut microbiota composition. Therefore, we identified mucosa-associated microbiota composition of the ileum and cecum at 10 and 30 wk of age. At 30 wk of age, we further identified luminal microbiota composition from combined content of the ileum, ceca, and colon. FP phenotypes could not be distinguished from each other in mucosa-associated or luminal microbiota composition. However, HFP neutrals were characterized by a higher relative abundance of genera of Clostridiales, but lower relative abundance of Lactobacillus for the luminal microbiota composition compared to LFP phenotypes. Furthermore, HFP neutrals had a higher diversity and evenness for the luminal microbiota compared to LFP phenotypes. FP genotypes could not be distinguished from each other in mucosa-associated microbiota composition. Yet, FP genotypes could be distinguished from each other in luminal microbiota composition. HFP birds were characterized by a higher relative abundance of genera of Clostridiales, but lower relative abundance of Staphylococcus and Lactobacillus compared to LFP birds. Furthermore, HFP birds had a higher diversity and evenness for both cecal mucosa-associated and luminal microbiota compared to LFP birds at adult age. In conclusion, we here show that divergent selection on FP can (in)directly affect luminal microbiota composition. Whether differences in microbiota composition are causal to FP or a consequence of FP remains to be elucidated.

Chicken lines divergently selected on feather pecking differ in immune characteristics.

It is crucial to identify whether relations between immune characteristics and damaging behaviors in production animals exist, as these behaviors reduce animal welfare and productivity. Feather pecking (FP) is a damaging behavior in chickens, which involves hens pecking and pulling at feathers of conspecifics. To further identify relationships between the immune system and FP we characterized high FP (HFP) and low FP (LFP) selection lines with regard to nitric oxide (NO) production by monocytes, specific antibody (SpAb) titers, natural (auto)antibody (N(A)Ab) titers and immune cell subsets. NO production by monocytes was measured as indicator for innate pro-inflammatory immune functioning, SpAb titers were measured as part of the adaptive immune system and N(A)Ab titers were measured as they play an essential role in both innate and adaptive immunity. Immune cell subsets were measured to identify whether differences in immune characteristics were reflected by differences in the relative abundance of immune cell subsets. Divergent selection on FP affected NO production by monocytes, SpAb and N(A)Ab titers, but did not affect immune cell subsets. The HFP line showed higher NO production by monocytes and higher IgG N(A)Ab titers compared to the LFP line. Furthermore the HFP line tended to have lower IgM NAAb titers, but higher IgM and IgG SpAb titers compared to the LFP line. Thus, divergent selection on FP affects the innate and adaptive immune system, where the HFP line seems to have a more responsive immune system compared to the LFP line. Although causation cannot be established in the present study, it is clear that relationships between the immune system and FP exist. Therefore, it is important to take these relationships into account when selecting on behavioral or immunological traits.

Effects of early feeding and dietary interventions on development of lymphoid organs and immune competence in neonatal chickens: A review.

With the ongoing intensification of the poultry industry and the continuous need to control pathogens, there is a critical need to extend our understanding of the avian immune system and the role of nutritional interventions on development of immune competence in neonatal chicks. In this review, we will focus on the ontogeny of the lymphoid organs during embryonic life and the first 2 weeks post-hatch, and how early feeding practices improve heath and modulate the development and function of the immune system in young chicks. The evidence for the positive impact of the nutrition of breeder hens on embryonic development and on the survival and immunity of their chicks will also be outlined. Additionally, we will discuss the vital role of supplemental feeding either in ovo or immediately post-hatch in chick health and immunity and the importance of these approaches in ameliorating immune system functions of heat-stressed chicks. To conclude, we provide some perspectives on a number of key issues, concerning the mechanisms of nutritional modulation of immunity, that need to be addressed. A thorough investigation of these mechanisms may assist in the formulation of diets to improve the immunity and general health status.

Plant expression of chicken secretory antibodies derived from combinatorial libraries.

Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes.

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.

Different levels of natural antibodies in chickens divergently selected for specific antibody responses.

We studied the presence of Natural antibodies in plasma samples from individual birds from selected chicken lines at young and old age. Binding, specificity, and relative affinity to various antigens were determined in plasma from non-immunized female chickens at 5 weeks of age, and in plasma obtained from the same chickens one year later using indirect two-step ELISA. Birds were from three different lines. The lines were divergently selected for either high (H line) or low (L line) antibody titers to Sheep Red Blood Cells at 5 weeks of age, next to a random bred control (C line). Binding of plasma immunoglobulins (Ig) from all three lines was found with chicken-egg-white protein (CEP), ovalbumin (OVA), myoglobin (MYO), thyroglobulin (THYRO), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and transferrin (TRANS). Significantly higher binding to most antigens was found with plasma Ig from adult birds from the H line as compared to plasma Ig from the L line, whereas binding of plasma Ig from C-line birds was in between or similar to the H or L line, respectively. Binding of Ig to all antigens in all three lines was significantly higher in plasma obtained at one year of age as compared to plasma obtained at 5 weeks of age. A competitive ELISA with homologous and heterologous antigens was used for determining specificity of the antigen-binding antibodies. Nai;ve plasma samples were characterized by a broad binding to all antigens tested. Inhibition of binding to specific antigens was possible with a broad range of heterologous antigens, but highest competition of binding was obtained with homologous antigen. Both linear regression analysis of serial dilutions of the plasma Ig binding the antigens, as well as competitive ELISA with homologous antigen indicated that plasma Ig from the H line and plasma Ig from the L line had similar affinity characteristics to the antigens tested with the exception of OVA and KLH. Pooled non-immune plasma from H line birds bound to CEP, OVA, THYRO, TRANS, MYO, KLH, and salt-precipitated extracts and supernatants of extracts from chicken heart, spleen, liver, brain, bursa, thymus, and kidney, respectively, as determined by Western blotting. The increasing presence of antibodies in nai;ve chicken plasma binding heterologous and homologous (tissue) antigens indicates the presence of Natural antibodies in poultry. Apart from age, increasing levels of Natural antibodies may be related with the genetically based magnitude of specific antibody levels in the chicken lines studied.

Adoptive transfer of natural antibodies to non-immunized chickens affects subsequent antigen-specific humoral and cellular immune responses.

To determine a regulatory function of natural antibodies in the immune response of chickens, pooled plasma obtained from non-immunized (naïve) 15 months old hens was subjected to keyhole limpet hemocyanin (KLH) antigen-affinity chromatography. Purified KLH-binding antibodies were adoptively transferred intravenously to 5 weeks-old cocks that were subsequently immunized subcutaneously 24 h later with KLH. Control groups consisted of birds that were either adoptively transferred with KLH-binding antibodies purified from plasma of KLH-immunized chickens, or PBS, or a salt precipitated total immunoglobulin fraction obtained from the corresponding pooled nai;ve chicken plasma, respectively.Total, IgM and IgY antibody titers to KLH in the plasma of recipients adoptively transferred with KLH-NAb, but not in the plasma of the groups transferred with salt precipitate or KLH-binding specific antibodies, were significantly enhanced as compared to the non-treated, KLH immunized group. Titers of IgA antibodies binding KLH were decreased in the plasma of the group that received specific KLH-binding antibodies, but not in the plasma of the other groups. Proliferation from peripheral blood leucocytes in whole blood from the KLH-NAb treated group, the group treated with KLH-binding specific antibodies and the group treated with salt precipitate, respectively, to both concanavalin A and KLH were significantly decreased as compared to the group receiving PBS. Our data show that antigen-specific antibodies can be isolated from plasma obtained from non-immunized chickens. Such antibodies that resemble natural antibodies as described in mammals may perform an important role in the enhancement of subsequent antigen-specific antibody responses or the maturation of the immune system, which may differ from the role of specific antibodies.