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. Background - Selection for general immune response in poultry has been proposed as a sustainable alternative to selection for resistance against specific diseases, because progress with the selection for resistance approach might be hindered by interactions between host and pathogen which would lead to continuous adaptability on both sides. In addition, it would not be feasible to select for disease resistance against the tremendous number of different pathogens that an animal could face in his entire life cycle. Several general immune traits were experimentally selected for in chicken lines  revealing that the different immune response mechanisms may have different genetic components . This study was based on three lines of White Leghorn Chickens that have been selected for 12 generations for one of three different immune response traits, high antibody response (ND3), cell mediated activity (PHA) and phagocytic activity (CC). Line ND3-L was selected on ND3, line PHA-L was selected for PHA, and line CC-L for CC, but all lines were measured for all three traits. The fourth line was a contemporary random bred Control maintained throughout the selection experiment. The results of the selection have been described by Pinard van der Laan . Briefly, 200 chicks per line were hatched (800 chicks in total) in a single batch every year. Selection for each trait was done by within-family mass selection based on individual phenotype. Heritabilities estimated for the three selection criteria ND3, PHA and CC were 0.35, 0.13 and 0.15, respectively, and correlations between the traits were not significant . The assessment of the disease resistance capability of the selected lines is currently under investigation and must be completed before any transposition of the results of the present work to the industry may be developed. The question that arose from this long term selection experiment was to determine if the in-vivo selection had changed the level of other immune response traits, which is to test correlated effects, with the overall aim to investigate whether the selection was trait, antigen, mechanism or pathway specific. This might result in adding other antigens or mechanisms in the long term selection experiment. The second question was to determine whether the response for the three selection criteria had modified the levels of the humoral components of the innate immune system (natural antibodies). Finally, we were interested in estimating the associations between the immune response traits under artificial selection and the newly measured ones. First, we investigated if the selected lines differed in their immune capabilities to mount an immune response to two other complex T-cell dependent antigens: Keyhole Limpet hemocyanin (KLH) and Mycobacterium butyricum, respectively. KLH is a copper-containing high molecular weight protein, found in the sea mollusc Megathura crenulata, which is commonly used as a soluble model protein known to induce a TH-2 like response . Mycobacterium is a solubilized particulate antigen that induces a TH-1 response in rodents . Significant differences were found previously between high and low chicken lines selected for SRBC (Sheep Red Blood Cells), for antibody response to M. butyricum  and for KLH [6,7], and chickens from the high antibody response line showed higher titers, irrespective of the antigen. Secondly, the level of natural antibodies binding two different T-cell independent antigens, Lipopolysaccharide (LPS) and Lipoteichoic acid (LTA), were measured to identify if the selection had changed the levels of the humoral components of the innate immune system. Both LPS and LTA are cell wall components, and represent associated patterns of gram negative (LPS) and gram positive (LTA) bacteria, respectively. In addition, as a further measure of the innate immunity, natural anti-Gal antibodies were measured. These antibodies represent IgM antibodies binding surface carbohydrate structures shared by a variety of pathogens . Natural anti-¿-Gal antibodies were measured in plasma samples by rabbit agglutinin levels, indicating antibodies reactive with Gal ¿ 1-3Galß-1-4GlcNAc-R, otherwise identified as the ¿-gal epitope. Their presence in the avian species has been previously demonstrated . Chickens as well as primates lack the functional ¿1, 3-galactosyltransferase gene and consequently produce high levels of anti-¿-Gal antibodies in response to the colonisation of the intestinal micro flora with galactosil bearing bacteria [10,11]. Previous studies on lines divergently selected for antibody titers to SRBC, demonstrated positive and moderately high correlation between RRBC (rabbit red blood cell) and SRBC titers . Furthermore, a recent study reported equally high levels of anti-gal antibodies in bile of high and low diet-efficient hens . In the present work, a total of 400 birds from the 12th generation of selection were studied. Half of the birds were immunized with KLH and the other half with M. butyricum particles. Levels of Ab to all antigens were measured before and 7 or 11 days after immunization, and correlations between them were tested. Principal component analysis was performed on the measures of immune response to better visualize the association between the new measures and the three selected lines ND3-L, PHA-L and CC-L. Results - Group immunized with KLH (Group A) Mean values and results of the analyses of variance are listed in Table 1. As expected from the past selection, lines had different (P ¿ 0.001) mean values for the selected traits, but there were also significant line effects (P ¿ 0.05 to P ¿ 0.001) on four of the other measures of immunity.
- keyhole limpet hemocyanin
Minozzi, G., Parmentier, H. K., Mignon-Grasteau, S., Nieuwland, M. G. B., Bed'hom, B., Gourichon, D., Minvielle, F., & Pinard-van der Laan, M. H. (2008). Correlated effects of selection for immunity in White Leghorn chicken lines on natural antibodies and specific antibody responses to KLH and M. butyricum. BMC Genetics, 9, . https://doi.org/10.1186/1471-2156-9-5