Selective breeding for high natural antibody level increases resistance to avian pathogenic Escherichia coli (APEC) in chickens.

Keyhole limpet hemocyanin (KLH)-binding natural antibody (NAb) titers in chickens are heritable, and higher levels have previously been associated with a higher survival. This suggests that selective breeding for higher NAb levels might increase survival by means of improved general disease resistance. Chickens were divergently selected and bred for total NAb levels binding KLH at 16 weeks of age for six generations, resulting in a High NAb selection line and a Low NAb selection line. To for test differences in disease resistance, chickens were challenged with avian pathogenic Escherichia coli (APEC) in two separate experiments. Chickens at 8 days of age received one of four intratracheal inoculations of 0.2 mL phosphate buffered saline (PBS): 1) mock inoculate, 2) with 0.2 mL PBS containing 108.20 colony-forming units (CFU)/mL APEC, 3) with 0.2 mL PBS containing 106.64 CFU/mL APEC, and 4) with 0.2 mL PBS containing 107.55 CFU/mL APEC. Mortality was recorded during 7 days post inoculation. Overall, 50-60% reduced mortality was observed in the High line compared to the Low line for all APEC doses. In addition, morbidity was determined of the surviving chickens at 15 days of age. The High line had lower morbidity scores compared to the Low line. We conclude that selective breeding for high KLH-binding NAb levels at 16 weeks of age increase APEC resistance in early life. This study and previous studies support the hypothesis that KLH-binding NAb might be used as an indicator trait for to selective breed for general disease resistance in an antigen non-specific fashion.

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

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

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

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

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

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

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

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

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.

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.