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Record number 532211
Title Selective breeding on natural antibodies in chickens
Author(s) Berghof, Tom V.L.
Source Wageningen University. Promotor(en): Henk Bovenhuis; Bas Kemp, co-promotor(en): Henk Parmentier; Jan van der Poel. - Wageningen : Wageningen University - ISBN 9789463437257 - 199
Department(s) Animal Breeding and Genetics
Publication type Dissertation, internally prepared
Publication year 2018
Availibility Full text available from 2019-04-19

In modern poultry production, high numbers of birds are housed at high densities. Recent changes in production systems, and management have been implemented with additional challenges: the change from battery cages to free roaming systems, and the reduction in preventive use of antibiotics have increased the risk of diseases, and disease spreading. The costs of diseases in poultry production are considerable, and several disease intervention strategies are currently used, or investigated. One potential strategy is to selectively breed chickens for increased general disease resistance. However, general disease resistance is difficult to define. Instead, selective breeding could be done on one, or several indicator traits, that are related to resistance against multiple pathogens. In addition, indicator traits should be heritable, and easy, and cheap to measure. Natural antibodies (NAb) might be a suitable indicator trait for selective breeding for general disease resistance. NAb are antigen-binding antibodies present in healthy individuals without previous exposure to the recognized antigen. NAb have a diverse range of functional roles: they play a role in maintaining homeostasis/housekeeping, regulation of the immune system, preventing auto-immunity, and increasing disease resistance. Moreover, NAb levels binding keyhole limpet hemocyanin (KLH) around adolescence were previously associated with survival in layer chickens, and were estimated to be heritable. Therefore selective breeding on KLH-binding NAb levels around adolescence might be a promising strategy to increase general disease resistance of layer chickens.

The objectives of this PhD thesis were:

1) to investigate the genetic variation of KLH-binding NAb levels in adolescent layer chickens;

2) to investigate the potential of KLH-binding NAb levels as an indicator trait for general disease resistance by

a) divergently selective breeding on total KLH-binding NAb titers, and

b) inoculating these NAb selection lines with avian pathogenic Escherichia coli (APEC); and

3) to investigate possible correlated selection responses on the immune system, and on production traits.

1) To investigate the genetic variation of KLH-binding NAb levels in adolescent layer chickens, heritabilities were estimated, and genome-wide association studies (GWAS) were performed.

Heritabilities were estimated in the base population of the selection experiment: 3,689 white purebred laying chickens were phenotyped around 16 weeks of age for total KLH-binding NAb titers, and for the isotypes IgM, IgA, and IgG. Heritabilities were 0.12 for total
KLH-binding NAb titers, 0.14 for IgM, 0.10 for IgA, and 0.07 for IgG. This means that
KLH-binding NAb are heritable, and selective breeding is possible. In addition, high positive genetic correlations were estimated, which means that selective breeding for one NAb type (i.e. total KLH-binding NAb titers) will result in similar responses of the other NAb (iso)types.

The GWAS were performed with 57,636 single nucleotide polymorphisms (SNP) for total KLH-binding NAb titers, and for the isotypes IgM, IgA, and IgG on 1,628 white purebred laying chickens of the same line as the base population. One genomic region was significantly associated to KLH-binding IgM NAb titers, and to a lesser extent to total KLH-binding NAb titers. The region showed full dominance, and had a major effect on IgM. This region is located on chromosome 4, and contained two Toll-like receptors (TLR). To further characterize the found association, total antibody concentration, and antibody concentrations of the isotypes IgM, IgA, and IgG were measured as well, and GWAS were performed. One genomic region, the same as identified before, was significantly associated to IgM antibody concentration. Full sequence data of key ancestors of the study population allowed imputation to full genome sequence for further association: 16 candidate genes were identified. SNP located in coding regions of these candidate genes were checked for predicted changes in protein functioning. One SNP, a C/G polymorphism at 69,965,939 base pairs (Gallus_gallus-5.0), received the maximum impact score from two independent prediction tools, which makes this SNP the most likely causal variant. The C-variant had an allele frequency of 0.45, showed a dominant mode of gene action, and was associated with high IgM levels. The G-variant had an allele frequency of 0.55, and was associated with low IgM levels. This SNP is located in TLR1A, which suggests a fundamental role of TLR1A on regulation of IgM levels, or B cells, or both.

2) To investigate the potential of KLH-binding NAb levels as an indicator trait for general disease resistance, a) layer chickens (originating from the base population) were divergently selectively bred on total KLH-binding NAb titers, and b) two generations of these NAb selection lines were inoculated with avian pathogenic Escherichia coli (APEC).

a) The selection criterion was total KLH-binding NAb titers at 16 weeks of age, and selection was based on own performance (i.e. mass selection). Chickens of the base population were selected to either breed the High NAb selection line (High line), or the Low NAb selection line (Low line). Selective breeding was performed for 6 generations. Each generation consisted of approximately 600 chickens per line. The average genetic differences in KLH-binding NAb titers at 16 weeks of age increased per generation with 0.36 for total NAb titers (selection criterion), 0.40 for IgM, and 0.32 for IgG (based on estimated breeding values (EBV)). Selective breeding on total KLH-binding NAb titers at 16 weeks of age was therefore proven to be possible.

Interestingly, the genetic progression of average titers in the High line reduced for
KLH-binding IgM NAb titers from generation 4 to generation 6, but was unaffected in the Low line. This could be the result of the dominance effect of the TLR1A variants present: in generation 6, the High line females had a C-variant frequency of 0.64, and a G-variant frequency of 0.36, and the Low line females had a C-variant frequency of 0.08, and a
G-variant frequency of 0.92. The amount of additive genetic variance, based on the TLR1A variants present in the NAb selection lines, was lower in the High line compared to the Low line: i.e. genetic improvement for higher KLH-binding IgM NAb levels was more difficult compared to genetic improvement for lower KLH-binding IgM NAb levels. Nevertheless, sufficient variation remains present to continue divergent selective breeding on NAb levels, even after six generations of selection.

b) Selective breeding for KLH-binding NAb levels does not necessarily result in differences in general disease resistance. Therefore the NAb selection lines were inoculated with avian pathogenic Escherichia coli (APEC). APEC is an opportunistic pathogen, mostly found in the respiratory tract. APEC causes colibacillosis, and can eventually lead to death. It has several antibiotic resistant mechanisms, and vaccination is not sufficiently protective. APEC is therefore a relevant poultry disease to consider. Generation 4, and generation 6 were intratracheally inoculated with one of three doses of APEC at 8 days of age. Mortality was recorded during 7 days, after which the experiment was ended. The observed mortality for all APEC doses was 2 to 3 times higher in the Low line compared to the High line. In addition, the surviving chickens at 15 days of age of the High line appeared to be less influenced by the infection compared to the surviving chickens of the Low line: morbidity scores of colibacillosis were lower, and body weight, and relative organ weights were higher. However, the exact protective mechanism, that underlies APEC resistance, remains to be identified.

3) To investigate possible correlated selection responses on the immune system, and on production traits, several traits were measured at different ages in several generations.

The NAb selection lines were observed for a diverse set of immunological traits at multiple ages during the selection experiment. The High NAb line had, compared to the Low line, higher KLH-binding NAb levels, and other antigen-binding NAb levels in general, a higher specific antibody response against human serum albumin (HuSA), higher antibody concentration, more peripheral B cells, and thrombocytes (percentages), a higher bursa weight, and a higher spleen weight. No line differences were observed for specific antibody responses against KLH, and avian tuberculin purified protein derivative of Mycobacterium avium (PPD), peripheral T cells, γδ T cells, NK cells, and antigen-presenting cells (percentages), and liver weight. This suggests that KLH-binding NAb selection has positive correlated selection responses for most immune traits, and no negative correlated responses (for the measured traits).

Several production traits were observed in relation to (selection on) KLH-binding NAb levels. Correlations between KLH-binding NAb types, and several production traits were estimated based on 2,385 females of the unselected base population. A significant positive genetic correlation was found between KLH-binding IgG NAb titers, and the feed conversion ratio (FCR; consumed feed/egg mass produced) ( ). In addition, several significant, though small phenotypic correlations ( ) were found between KLH-binding NAb levels, and some production traits. In the NAb selection lines, monthly body weight measurements suggest that the High line has a higher growth curve compared to the Low line. However, the adult body weight did not differ between the NAb selection lines. In generation 5, selected females (35 weeks of age) were monitored for production for three weeks: the High line had a higher egg weight, and whiter eggshells compared to the Low line. The NAb selection lines were equal for eggshell thickness, eggshell breaking strength, and FCR. In general, production traits did not seem to be (strongly) negatively affected by NAb selection, which gives room for simultaneous improvement of KLH-binding NAb levels, and production traits.

Given available studies in literature, and this PhD thesis, I hypothesize that KLH-binding IgM NAb are a proxy for the humoral adaptive baseline immunity. KLH-binding IgM NAb show the potential of an individual’s humoral adaptive immune system. This means that not necessarily KLH-binding NAb, but the IgM concentration, or, more likely, the number of naive, resting, IgM-only B cells present in an individual, may determine general disease resistance.

In conclusion, selective breeding for total KLH-binding NAb levels around adolescence in chickens is feasible, and influences APEC resistance at young age. Given available studies in literature, and this PhD thesis, I hypothesize that selective breeding for KLH-binding NAb levels increases bacterial disease resistance, without detrimental effects on the immune system, and production traits. However, more research (e.g. infection experiments with different pathogens, or field experiments) in the NAb selection lines, and in other chicken lines, or chicken breeds, is required to confirm this, and to investigate increased general disease resistance.

All comments Post a comment said...

Dear visitor,


My PhD thesis will be under embargo for some time. However, I can send you a digital/printed copy if you like. Please contact me at .


Kind regards,


Tom Berghof

2018-01-24   14:02:07
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