Genetic Bases for Resistance and Immunity to Avian Diseases

<p>NON-TECHNICAL SUMMARY:<br/> Poultry production creates high value in the U. S. economy. Direct and indirect effects of disease and ineffective immune responses reduce profits. This proposal is intended to investigate genes crucial to immunity by comparing the genes expressed during embryonic development of immune tissue and during the course of an immune response in two pair of chicken lines divergently selected for high or low antibody responses. Improving the immune response would significantly reduce the production costs attributable to disease, medications, and increased labor cost while improving food safety as well as quality.
<p>APPROACH:<br/> Compare immune-related gene expression during lymphoid tissue development in selected high and low antibody lines. In the previous project period, primary and secondary immune tissues (thymus, bursa of Fabricius, spleen) were collected at incubation days 15, 18 and 21 of development from the Virginia Tech high antibody (HAS) or low antibody (LAS) lines. Tissue samples remain preserved in RNAlater for subsequent RNA extraction. Additional High (HA) and Low (LA) antibody lines eggs for sampling will be obtained from my N. C. State University collaborator, Dr. Chris Ashwell. Standard temperature and humidity incubation will occur at the University of New Hampshire. Tissue samples from the thymus, bursa and spleen will be collected from five embryos of each line (HAS, LAS) at the stated time intervals (15, 18, 21 days incubation). RNAlater (Applied
Biosystems-Ambion, Foster City, CA) will be used for 80?C storage of individual tissue samples according to the company recommended protocol. Eggs from the Wageningen high (WH) and low (WL) antibody lines will be incubated and sampled as described in the paragraph above. This will be supplied by another collaborator, Dr. Henk Parmentier at Wageningen University, The Netherlands. Significant obstacles hinder importation of eggs into the United States. These limitations can be overcome if the samples collected are preserved in RNAlater. The Wageningen lab has agreed to supply these samples as part of the collaboration. The samples from the four selected lines, two high antibody (HAS, WH) and two low antibody (HAS, WH), will be compared for gene expression. Extraction of total RNA from five samples of each tissue per line use the method described by Druyan and associates (2008).
Briefly, acidified phenol-chloroform-isoamyl alcohol (125:24:1) will be used to isolate the RNA from 100 mg of homogenized tissue. The mixtures are centrifuged to separate the phases followed by an additional (49:1) cholorform-isoamyl alcohol extraction of the aqueous phase. RNA will then be precipitated from the upper aqueous phase of extraction 2 using isopropanol for two hours. The precipitated RNA is pelleted via centrifugation, washed with 75% ethanol and dissolved in DEPC (diethylpyrocarbonate) water. Gel electrophoresis and 260 nm spectrophotometric absorbance will be applied to test the isolated RNA for quality and concentration. The RNA purity will seek an A260:280 ratio >1.85. Tissue RNA from the bursa, thymus and spleen will be compared within that particular tissue and time point. In addition, comparisons will be made for each tissue type within lines (HAS vs LAS, WH vs
WL) and across lines (HAS vs WH, LAS vs WL). Fluorescent dyes Cy-3 and Cy-5 will be used to label the cDNA. To permit a dye-swap to detect potential bias in labeling, aliquots of each sample will be labeled with both dyes. The North Carolina State University Poultry Genomics Laboratory has produced multiple focused microarrays targeting chicken genes in particular physiological states. The focused microarray for these studies employs genes associated with the immune system. Using chicken data archived in the Gene Index Project (http://compbio.dfci.harvard.edu/tgi/tgipage.html), 320 genes representing the chicken immune response were selected for the array. Seventy bp oligonucleotides were synthesized. The individual oligonucleotides occur 12 times in the same array. The arrangement involved spotting each oligonucleotide 3 consecutive times and repeated four times on the entire array.
Small variations in gene expression are more-easily detected with this configuration. New genes are added to the array as needed and other arrays focused in additional systems have been created. The microarrays will be scanned with a ScanArray GX PLUS Microarray Scanner. The scan data will be processed using JMP Genomics from SAS. Normalization and weighted regression with smoothing will be applied to the initial data that is log2 transformed (Druyan et al., 2008). Analysis of variance using a mixed-model can then be used to calculate the P values. Significance will be at the P < 0.05 (Hochberg, 1988) with a Bonferroni correction. Four replicates per slide for each gene represented by an oligonucleotide will result from the three consecutive spots averaged. Genes with significant differential expression will be examined using the Metacore database (GeneGo, Inc., Carlsbad, CA) for
pathway and process analysis. Nikolsky and co-workers (2005) described the utilization of the annotated database. Ranking the genes by their differential expression reveals the biological processes associated with the analysis. Furthermore, Metacore examines the ranked list of differentially-expressed genes though shortest path of significant gene interaction algorithms to uncover important biological networks. Compare gene expression during an immune response to the selection antigen. In order to understand the origin of the differential immune response, birds from each of the selected pairs will be injected with SRBC, the antigen used in the selection for high or low antibody response. Fifty birds from each of the two high antibody (HAS, WH) and two low antibody (HAS, WH) will be injected intravenously with 1 milliliter of 1% SRBC. This is the route of injection used for selection in
the HAS and LAS lines from Virginia Tech. Spleen samples will be collected in RNAlater at prior to injection (time 0) and 6, 12, 18 and 24 hours after injection. Additional samples will be collected from 10 birds at 1, 2, 3, 4, and 5 days post injection. These sampling periods will allow expression measurements in the early phase after antigen injection as well as the period up to the peak antibody response (Zhao et al, 2012). RNA will be extracted from the spleen samples and reverse transcribed to cDNA as described in the previous section. Labeling of the cDNA and application to the immune focused microarray wil proceed as described above. Analysis of the data will be done with JMP Genomics (SAS Institute Inc., Cary, NC) as described. The gene expression comparisons of interest will be HAS vs LAS, WH vs WL, HAS vs WH and LAS vs WL. This study will narrow the range of genes that are
important for a high or low antibody response. Significant differentially-expressed genes will be queried in the Metacore database to reveal important pathways and biological networks in the particular response. Fifty birds from each of the two high antibody (HAS, WH) and two low antibody (HAS, WH) will be injected intramuscularly with 1 milliliter of 1% SRBC. This is the route of injection used for selection in the WH and WL lines from Wageningen University. Sample collection and analytical procedures will be identical to those in part one. The gene expression comparisons of interest will be HAS vs LAS, WH vs WL, HAS vs WH and LAS vs WL. The gene with differential expression will point to critical regions and pathways that produce higher or lower antibody responses. Portions of all samples from all three studies will be held in RNAlater. At a subsequent time when costs have reduced
significantly or other scientists are interested, the samples can be processed for RNAseq which will enhance the data obtained. Current costs, capitol and human, are still too great to be done in these studies.