USING MICROARRAYS TO TEST THE LINK BETWEEN COLORATION AND HERITABLE DISEASE RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0217294
• Project No.: ALA021-1-09002
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2014

Project Director
Hill, G.

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Biological Sciences

Non Technical Summary
One of the most intriguing ideas in evolutionary biology is the suggestions that sexually reproducing animals choose mates who carry "good genes" for survival. Choosing mates with such good genes would enhance the genetic quality of offspring. Further it is proposed that ornamental traits such as the antlers of deer or the bright coloration of feathers might serve as indicators of genetic quality. This good genes hypothesis is very intuitive and appealing but it has proven difficult to test. New genetic tools, however, provide an opportunity to test the theory. We propose to use new genetic tools for studying gene expression to identify genes that appear to function in the immune response of the House Finch when it is infected by the bacterium Mycoplasma gallicepticum (MG). Once these candidate genes are identified we will use another set of genetic tools to search for genetic variation that could be inherited and could be the "good genes" sought by choosing females. Finally we will test the good genes hypothesis by correlating the brightness of red plumage in male house finches to their genetic makeup, looking for associations between good genes and color display. This research is aimed at improving our basic understanding of immunogentics and the evolution of disease resistance in animals. There are potential direct benefits for agriculture. The focal pathogen in this study, MG, is among the most damaging diseases to U.S. poultry, costing millions of dollars in treatment and lost sales. A better understanding of the co-evolution of MG and a novel bird host and how evolution shapes immunity to MG will allow for the development of better treatments for this important poultry disease.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	0820	1040	25%
304	4010	1040	25%
311	0820	1040	25%
311	4010	1040	25%

Knowledge Area
311 - Animal Diseases; 304 - Animal Genome;

Subject Of Investigation
4010 - Bacteria; 0820 - Wild birds;

Field Of Science
1040 - Molecular biology;

Keywords

disease resistance

microarray

good genes

mycoplasma

poultry

house finch

gene expression

immunogenetics

Goals / Objectives
OBJECTIVES: The primary goal of this project is to test the hypothesis that ornamental traits signal the genetic quality of individuals and hence that sexual selection can drive the evolution of genetic disease resistance. This project addresses a key conceptual issue in evolutionary biology and holds the potential to have a significant impact on key evolutionary theory. The proposed research also links to a number of practical issues related to agriculture. The focal pathogen in this study, Mycoplasma gallicepticum, is among the most damaging pathogens to U.S. poultry, costing millions of dollars in treatment and lost sales. A primary goal of this research is to gain better understanding of the co-evolution of Mycoplasma gallicepticum and a novel avian host and how evolution shapes immunity to Mycoplasma gallicepticum. This research is directed toward providing critical basic information for reducing the impact of Mycoplasma gallicepticum on the poultry industry. Using comparative genomics methods, we have identified several genes for immunity in House Finches that map directly onto genes for immunity in chickens, including several MHC class II associated genes, a T-cell receptor, a lymphocyte cytosolic protein, and several immunoglobulins. A final object of this study is to expand our understanding of genetic control of immune responsiveness to Mycoplasma gallicepticum in avian systems. This will provide basic information about how birds respond to novel pathogens and how those pathogens in turn counteract avian defenses. This will have direct benefits for the poultry industry by expansion of our understanding of how the avian immune system responds to pathogens, and thus provide the foundation for better strategies for combating novel pathogens affecting the poultry industry.

Project Methods
Infection experiments We will bring 175 finches to Auburn and randomly divide the birds into four flocks. The control (non-infected flock) will consist of 25 birds. The three experimental flocks infected with MG (see below) will consist of 50 birds each. We will have three levels of analyses in our infection experiments: 1) comparing gene expression between House Finches that have been infected with MG versus not infected, 2) within infected birds, comparing maximum symptoms at days 3 and 14 as a measure of resistance to gene expression, 3) within infected birds, comparing symptoms at day 21 as a measure of disease recovery to gene expression. To study gene expression related to resistance and recovery of MG, we will sacrifice males in the three infection groups at 3, 14, and 21 days. We will then compare gene expression in males in each of these groups to gene expression in birds that are not infected. We will then compare gene expression between the 20 males with the worst symptoms (least able to resist/recover) to the 20 males with the least symptoms (most able to resist/recover). House Finch microarray We will build a microarray using the cDNA sequences generated by the 454 transcriptome sequencing described above. Multiplex RT-PCR We will use the findings from this genome-scale microarray to select candidate genes for more detailed investigation. Specifically, we will select differentially expressed genes known or suspected to be involved in immune response for incorporation into multiplexed RT-PCR reactions. We will select a set of between 20-30 genes that were differentially expressed for each timepoint that tissue was collected during the aviary infection experiments (3 days, 14 days, and 21 days). Thus, we will create 3 separate multiplexes - one for each timepoint. We will then compare each individual bird's ability to resist or recover from infection (as determined by physical symptoms, levels of antibody production, and viremia titers) with expression levels of genes in the multiplex. Discovering sequence differences between resistant and susceptible birds Our macroarray (Wang et al. 2006) and microarray studies have already identified two genes that are known to be strongly upregulated during House Finch infection with MG. The sequences of coding regions of these genes are already known from our previous study and further such sequences will be discovered through our transcriptome study. We expect to be able to study the sequence variation in approximately 30 candidate genes and their flanking regulatory sequences, using techniques that we have already demonstrated. We will quantify the hue, chroma, and brightness of the central upper breast of 300 male House Finches. We will identify 25% of males (75 individuals) with the drabbest plumage and 25% of males (75 individuals) with the brightest plumage, and genotype these 150 males using blood samples collected at capture.

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: One of the most intriguing ideas in evolutionary biology is the suggestions that sexually reproducing animals choose mates who carry "good genes" for survival. Choosing mates with such good genes would enhance the genetic quality of offspring. Further it is proposed that ornamental traits such as the antlers of deer or the bright coloration of feathers might serve as indicators of genetic quality. This good genes hypothesis is very intuitive and appealing but it has proven difficult to test. New genetic tools, however, provide an opportunity to test the theory. We propose to use new genetic tools for studying gene expression to identify genes that appear to function in the immune response of the House Finch when it is infected by the bacterium Mycoplasma gallicepticum (MG). Once these candidate genes are identified we will use another set of genetic tools to search for genetic variation that could be inherited and could be the "good genes" sought by choosing females. Finally we will test the good genes hypothesis by correlating the brightness of red plumage in male house finches to their genetic makeup, looking for associations between good genes and color display. This research is aimed at improving our basic understanding of immunogentics and the evolution of disease resistance in animals. There are potential direct benefits for agriculture. The focal pathogen in this study, MG, is among the most damaging diseases to U.S. poultry, costing millions of dollars in treatment and lost sales. A better understanding of the co-evolution of MG and a novel bird host and how evolution shapes immunity to MG will allow for the development of better treatments for this important poultry disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has involved 3 doctoral students and 4 undergraduate students at Auburn University. How have the results been disseminated to communities of interest? Talks were presented at the 2013, 2014, and 2015 meetings for the Society for Comparative and Integrative Biology. Graduate student Molly Staley was the lead and presenting author and Dr. Geoffrey Hill was the second author for three talks, and undergraduate student Hillary Rizk was the lead and presenting author on a fourth presentation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In September through Nov 2014 we conducted infection experiments with leghorn chickens. 75 five-week-old leghorns were received from a dealer and allowed to acclimate to cages in a quarantine facility at the school of veterinary medicine at Auburn University. After 10 days of acclimation, chickens were divided into 5 treatment groups with 15 birds per treatment. These treatments were: 1) infection with House Finch Strain of MG and incubate for 4 days; 2) nfection with House Finch Strain of MG and incubate for 14 days; 3) infection with chicken Strain of MG and incubate for 4 days; 4) nfection with chicken Strain of MG and incubate for 14 days; 5) sham manipulated control. At the designated number of days, all chickens were euthanized and spleen and trachea tissue was collected. There spleen and trachea samples are currently being analyzed to study the effects that an adapted versus a non-adapted pathogen have on immune responsiveness of host tissues.

Publications

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: One of the most intriguing ideas in evolutionary biology is the suggestions that sexually reproducing animals choose mates who carry "good genes" for survival. Choosing mates with such good genes would enhance the genetic quality of offspring. Further it is proposed that ornamental traits such as the antlers of deer or the bright coloration of feathers might serve as indicators of genetic quality. This good genes hypothesis is very intuitive and appealing but it has proven difficult to test. New genetic tools, however, provide an opportunity to test the theory. We propose to use new genetic tools for studying gene expression to identify genes that appear to function in the immune response of the House Finch when it is infected by the bacterium Mycoplasma gallicepticum (MG). Once these candidate genes are identified we will use another set of genetic tools to search for genetic variation that could be inherited and could be the "good genes" sought by choosing females. Finally we will test the good genes hypothesis by correlating the brightness of red plumage in male house finches to their genetic makeup, looking for associations between good genes and color display. This research is aimed at improving our basic understanding of immunogentics and the evolution of disease resistance in animals. There are potential direct benefits for agriculture. The focal pathogen in this study, MG, is among the most damaging diseases to U.S. poultry, costing millions of dollars in treatment and lost sales. A better understanding of the co-evolution of MG and a novel bird host and how evolution shapes immunity to MG will allow for the development of better treatments for this important poultry disease. Changes/Problems: no major changes What opportunities for training and professional development has the project provided? This project has involved 3 doctoral students and 4 undergraduate students at Auburn university. How have the results been disseminated to communities of interest? Talks were presented at the 2013, 2014, and 2015 meetings for the Society for Comparative and Integrative Biology. Graduate student Molly Staley was the lead and presenting author and Dr. Geoffrey Hill was the second author for three talks, and undergraduate student Hillary Rizk wasthe lead and presenting author on a fourth presentation. What do you plan to do during the next reporting period to accomplish the goals? We are currently analyzing the tissue samples from infection experiments.

Impacts
What was accomplished under these goals? In September through Nov 2014 we conducted infection experiments with leghorn chickens. 75 five-week-old leghorns were received from a dealer and allowed to acclimate to cages in a quarantine facility at the school of veterinary medicine at Auburn University. After 10 days of acclimation, chickens were divided into 5 treatment groups with 15 birds per treatment. These treatments were: 1) infection with House Finch Strain of MG and incubate for 4 days; 2) nfection with House Finch Strain of MG and incubate for 14 days; 3) infection with chicken Strain of MG and incubate for 4 days; 4) nfection with chicken Strain of MG and incubate for 14 days; 5) sham manipulated control. At the designated number of days, all chickens were euthanized and spleen and trachea tissue was collected. There spleen and trachea samples are currently being analyzed to study the effects that an adapted versus a non-adapted pathogen have on immune responsiveness of host tissues.

Publications

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: This report is aimed primarily at professional agricultural research scientists, and secondarily at poultry farmers and veterinarians who address poultry diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has invovled 3 doctoral students and 3 undergraduate students at Auburn university. How have the results been disseminated to communities of interest? A talk was presented at the 2013 meeting for the Society for Comparative and Integrative Biology. Graduate student Molly Staley was the lead and presenting author and Dr. Geoffrey Hill was the second authoer. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period we will complete the second and final portion of the chicken infection experiments. Once we have all tissues collected, we will assess whether damage is due to host immune responses by comparing tracheal and conjunctiva tissue damage in chickens infected with their natural live MG strain versus natural heat-inactivated MG strain. Following euthanization of birds, we will formalin-fix tissues and then submit tissues to the Auburn University Histopathology Laboratory to be paraffin embedded, sectioned, and stained for microscopy work. we will then quantify tissue lesions and measure tracheal mucosal thickness following the methods of Nunoya et al (1987). Alternatively, MG products could cause direct damage to host tissues. We will compare tracheal primary cell cultures from chickens inoculated with house finch MG or chicken MG, respectively. We will then compare cultures inoculated with MG relative to controls for changes in cellular morphology and tracheal cell death. We predict that if virulence is primarily due to the host immune responses, then chickens treated with heat-inactivated MG will show similar levels of disease and tissue damage to those treated with live MG. We also predict there will be no difference between cell culture treatments.

Impacts
What was accomplished under these goals? In September 2013 we initiated infection experiments with leghorn chickens. 30 five-week-old leghorns were received from a dealer and allowed to acclimate to cages in a quarantine facility at the school of veterinary medicine at Auburn University. After 10 days of acclimation, fifteen leghorns were infected with a house finch strain of Mycoplasma gallisepticum and fifteen were sham inoculated. All birds were sacrificed on day 10 following inoculation and spleen and trachea tissue was collected. There spleen and trachea samples will be analyzed to study the effects that an adapted versus a non-adapted pathogen have on immune responsiveness of host tissues.

Publications

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: This is a new project and I am in the process of preparing for spring infection experiments that will comprise the core activity of this grant. I am currently obtaining a chicken strain of MG and I am verifying the viability of our House Finch MG culture. This project has been refocused to be more in line with the objectives of the Hatch program. I am now focusing on understanding the pathogenicity of a major poultry pathogen. The title of the project is now: The genetic basis for attuation of virulence of Mycoplasma gallisepticum in chickens following host shift PARTICIPANTS: Molly Staley--Doctoral student Roy Ge -- Doctoral student Hilary Rizk -- undergrad student Rosana Garcia -- Undergraduate student TARGET AUDIENCES: Our target audience is research specialists in agriculature as well as scientists studying host-parasite co-evolution. Our goal is to discover basic properties of mycoplasma that affect pathogenicity so products can be developed that tangibly improve US agriculture. PROJECT MODIFICATIONS: This project has been refocused to be more in line with the objectives of the Hatch program. I am now focusing on understanding the pathogenicity of a major poultry pathogen. The title of the project is now: The genetic basis for attuation of virulence of Mycoplasma gallisepticum in chickens following host shift

Impacts
This is a new research endeavor so no specific results have emerged yet.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: not funded. no outputs PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
not funded. no outcomes

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: project not funded; no output PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
project not funded; no output

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Not funded so still in planning phase PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Not funded so still in planning phase

Publications

• No publications reported this period