Senior Scientist Helle R. Juul-Madsen (Helle.JuulMadsen@agrsci.dk)
The vision is to develop a new genetic selection technology based on an effective innate immune response for production of chickens with polyvalent protection against diverse pathogens and increased livability. This will be achieved by identification of important genes and genetic markers in an old Danish chicken line (Hellevad) using modern biotechnology tools including genome-wide association studies.
One single gene Mannose Binding Lectin (MBL) has already been identified as a potential selection marker in this chicken line. Application of high-throughput genotyping based on Sequenom technology will enable the discrimination of chicken MBL genotypes. MBL belongs to the innate part of the immune system and is a key genetic factor in innate polyvalent resistance.
MBL interacts with the acquired immune response which is why unraveling the interaction between MBL and the adaptive part of the immune system is a prerequisite before an integrated MBL selection approach can be applied in the poultry industry where disease protection currently relies on wide usage of vaccinations.
The overall success criteria are:
· That the genetic basis of the robustness in the Hellevad chicken is characterized and developed into a genetic selection approach for breeding chickens.
· That the interaction between MBL and the adaptive immune response is revealed.
· That MBL genotypes determining low disease resistance of chickens are eliminated from Cobb populations.
This will increase animal welfare, food safety, the consumers’ accept, and the industry’s competitiveness.
Objectives of the project
The overall aim of the project is to create the necessary scientific basis for new breeding programs in commercial poultry production focusing on increased innate disease resistance. The following are specific project goals:
· To identify new genotypes in commercial chickens associated with polyvalent disease resistance.
· To determine the consequence of MBL deficiency in relation to susceptibility to Infectious Bronchitis Virus (IBV) and Salmonella using experimental infections.
· To determine the ability of MBL to stimulate/inhibit the adaptive part of the immune system and to discover new molecules that temporarily modulate the effect of MBL.
The project is supported by the Danish Council for Strategic Research, Danish Poultry Council, The Hatchery Hellevad, and Cobb-Vantress. Two PhD students and one post doc are attached to the project.
Status April 2013
Three steering group meetings have been held (February 2011, 2012 and 2013), where project results were scrutinized. Two biannual reports for each work package have been collected. The project has been presented frequently in the Danish and foreign press, primarily based on 2 press releases from Aarhus University and an article from Cobb-Vantress’ own magazine in 2011 and 2012.
WP1: DNA from the 2 parental lines of the Hellevad chicken has been tested for sex, pooled and sent for ’next generation sequencing’ by Cobb. The sequencing result will be compared with genomes from other egg laying chicken breeds by means of a 'selection sweep analysis' for identification of sites in the Hellevad genome that differ from other egg laying breeds and that may be associated with robustness.
WP2: The genotype frequencies in the 2 Hellevad parental lines have been determined. Only 3 haplotypes have been found in these lines of which none are associated with low concentration of MBL in serum. This supports our hypothesis that the natural selection having taken place for the past 50 years has made the Hellevad cross-breed particularly robust. Two of the haplotypes found differ statistically in their concentration of MBL in serum.
Together with the breeding company Cobb-Vantress we have had access to DNA and serum from 3 of their pure lines (Lines A, B and C). Nine alleles have been identified in the Cobb-animals, of which 6 alleles are identical to those found in Danish animals – including Hellevad. The haplotype A1, which causes low MBL in serum, has been found in a relatively high frequency in 1 of the 3 pure lines. Most alleles have consequently existed for very many years – maybe even since separation of chicken broilers and layers started.
For genotyping of the Cobb-animals we are developing a completely new genotyping tool KASPar which seems to be better suited than the Sequenome-platform. By typing 8 of the 15 found SNPs it is, in fact, possible to separate the 9 alleles that we have found. When the genotype frequencies have been completed, an association study in relation to the concentration of MBL in serum will be performed.
WP3: New immunological methods have been developed in the project. A qPCR method for measuring Infectious Bronchitis Virus (IBV) genomes has been established. A flow cytometric-based method for measuring leucocyte-subpopulations and a method for intracellular cytokine measurement have also been established.
During the first 2 years we have performed 6 experimental studies, 2 studies in relation to IBV and 4 studies in relation to Salmonella. Further, we have carried out an ontogeny study where selected immunological parameters have been followed in L10 animals from day-old chickens to egg layers as well as a study where the general immune competence in Cobb500 ’final product’ chickens has been investigated with a view to screening for other hereditary immunological selection markers than MBL (WP1).
WP4: MBL has been purified from chicken serum and used in binding studies. By means of flow cytometry we have shown that chicken MBL binds to salmonella-strains of the C1-serotype, which is known for containing many mannose units in their LPS. A 10-week-long ligand-vaccination study with IBV has been performed with L10. All data have been stored and the statistical data processing is currently being done. Three different MBL ligands (mannose, chitosan and fruit oligosaccharides (FOS)) have been tested resulting in marked differences in the animals’ vaccine response.
Generally, the project proceeds according to the estimated time schedule although the experimental studies have started late, which is why we consider trying to have the project prolonged by a year.
Kjærup, R.M., Skjødt K., Dalgaard T.S., and Juul-Madsen H.R. (2013). Chicken mannose-binding lectin 2 gene variants with influence on MBL serum concentration. Immunogenetics 65; 461-471. DOI: 10.1007/s00251-013-0689-6.
Juul-Madsen, HR, Norup, LR, Sørensen, P & Dalgaard, TS 2013, 'Ontogenic Development of Lymphocyte Subsets in Two Selected Chicken Lines that Differ in Mannose-Binding Lectin (MBL) Serum Concentration Under Conventional Rearing: SSI13-1030' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 268-268.
Ulrich-Lynge, SL, Dalgaard, TS, Norup, LR, Olsen, JE & Juul-Madsen, HR 2013, 'Colonization Resistance and Immunological Differences in Response to An Experimental Salmonella Infection between Two Chicken Lines Selectively Bred for High or Low Concentration of Mannonse-Binding Lectin: SSI13-1018' Scandinavian Journal of Immunology, vol 77, nr. 4, s. 274-274.
Kjærup, RM, Dalgaard, TS, Norup, LR, Sørensen P & Juul-Madsen, HR 2013, ’Can Vaccines be improved in Chickens by Adding Mannose-Bonding Lectin (MBL) Ligands?: SSI13-1101’ Scandinavian Journal of Immunology, vol 77, nr. 4, s. 320-320.