Integrative Functional and Physiological Genomics of Poultry

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3220	1040	30%
304	3299	1080	50%
311	3260	1090	20%

Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: Even though the Gallus gallus chromosome 16 (GGA 16) contains the major histocompatibility complex and is especially important in disease resistance in poultry, the gene map for GGA 16 is incomplete. Trisomy mapping using Cornell trisomy line chickens naturally exhibiting 2, 3 and 4 copies of GGA 16 is a powerful means for assigning genes to GGA 16. MHC-B, MHC-Y and CD1 genes were all previously assigned to GGA 16 by trisomy mapping. Here we combine trisomy mapping with array Comparative Genomic Hybridization (aCGH) to identify additional GGA 16 sequences among unassigned genomic sequence contigs. A custom 4x44K Agilent aCGH was developed that includes 40,000 probes with a spacing of about one 60mer probe for each 4Kb of unassigned genomic sequence. Also included were 500 GGA 16-specific positive control probes and 1,546 negative control probes specific for other autosomes and sex chromosomes. The DNA used in array hybridization included eight samples each from individuals within the Cornell trisomy line previously typed as disomic, trisomic, and tetrasomic for GGA 16. After scanning and feature extraction, data was normalized by scaling and centering methods. Significance was tested using mixed linear models comparing disomic individuals to tri- and tetrasomic individuals. The false discovery rate (FDR) was calculated. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of big concern to human health. The objective of this study was to identify proteins associated with AIV infections in two genetically distinct, highly inbred chicken lines that differ in AIV resistance (Fayoumi is moderately resistant and Leghorn is susceptible to AIV infection). Chickens were inoculated with 107 EID50 of H5N3 AIV or PBS at three weeks of age and lungs were collected at 4 days post infection. Proteins with equal amount of cytosolic, membrane, nuclear and cytoskeletal fractions were extracted by differential detergent fractionation followed by a Trichloroacetic acid/Acetone precipitation (total 8, 2 per line per treatment group). Proteins were then digested into peptides by trypsin and desalted using a peptide Macrotrap. Tryptic peptides were separated using multidimensional protein identification technology, which were subjected to data dependent MS/MS acquisition. Resulted MS/MS spectra were searched via Sequest against the chicken protein database in Ensembl. Fisher Exact test was used to determine significantly differentially expressed proteins between treatment groups or genetic lines. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: PD for this project has transferred to UC Davis.

Impacts
For CGH GGA16 mapping project, a total of 489 probes were found to be significant at a FDR of 10%, including 91 probes from unassigned contigs and 398 probes specific for GGA 16. These findings confirm our hypothesis that trisomy mapping and aCGH can be combined for assigning additional sequences to GGA 16 and have revealed a number of candidate genes for further study. For AIV project, there were 2742, 3049, 2002, and 2577 proteins identified in infected, and non-infected Fayoumi, and infected and non-infected Leghorn, respectively. Based on the cut-off P-value of 0.01 and fold-change of 2, there were 1408 and 612 proteins differentially expressed between infected and non-infected birds within Fayoumi and Leghorn lines, respectively. Between genetic lines, 1038 and 640 proteins were differentially expressed within infected and non-infected groups, respectively. More proteins had higher expression levels in Fayoumi than Leghorn birds pre-inoculation. Post infection more proteins highly expressed in Leghorn birds than in Fayoumi. Within genetic lines, more proteins were down-regulated in Fayoumi and more were up-regulated in Leghorn birds with AIV infection. Protein expression patterns changed with AIV infection in both lines, and the responses of these two lines were relatively opposite to each other. Differentially expressed proteins identified in this study could lead to new directions in the development of anti-viral drugs or vaccines in AIV infection in poultry.

Publications

Wang Y, N. Ghaffari, C. D. Johnson, U. M. Braga-Neto, H. Wang, R. Chen, H. Zhou. 2011 Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens. BMC Bioinformatics 12: Suppl 10, doi:10.1186/1471-2105-12-S10-S5.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, H. Zhou. 2011. Cecal transcriptome analysis of colonized and non-colonized chickens within two genetic lines that differ in cecal colonization by Campylobacter jejuni. Animal Genetics 42:491-500.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, F. McCarthy, S. Burgess, Y. Pevzner,H. Zhou. 2011. Systemic Response to Campylobacter jejuni Infection by Profiling Gene Transcription in the Spleens of Two Genetic Lines of Chickens. Immunogenetics in press.
Yu Y, J. Luo, A. Mitra, S. Chang, F. Tian, H. Zhang, P. Yuan, H. Zhou, J. Song. 2011 Temporal Transcriptome Changes Induced by MDV in Marek's Disease-Resistant and -Susceptible Inbred Chickens BMC Genomics 12:501.
Sandford E, M. Orr , E. Balfanz , N. Bowerman , X. Li , H. Zhou , T. J Johnson , S. Kariyawasam , P. Liu , L K Nolan and S. J Lamont. 2011. Spleen transcriptome response to infection with avian pathogenic Escherichia coli in broiler chickens. BMC Genomics 12:469.

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

Outputs
OUTPUTS: Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. The objective of this study was to identify microRNAs associated with AIV infections in chicken lung and to profile host response to AIV infection. Total RNA isolated from lung of low pathogenic H5N3 infected and uninfected broilers at 4 days post-infection was used for both microRNA deep sequencing and microarray analyses. Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial enteritis worldwide with poultry products one of the main sources of contamination. Transcriptome profiling of local cecum response to C. jejuni infection between two genetically distinct chicken lines (Line A is more resistant than line B) has been studied in our laboratory. To further understand the systemic molecular response mechanisms to a C. jejuni infection, day-old chickens from lines A and B were challenged orally with 1.8X10^5 colony forming units (cfu ) C. jejuni and the spleens were removed 7 d post-infection. Total RNA were isolated from the spleens and applied to a whole genome chicken microarray for a pair-comparison between infected (I) and non-infected (N) chickens within each line and between lines A and B. 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
For Avian influenza virus infection, a total of 3.2M and 3.4M filtered high quality reads were obtained from infected and non-infected chicken by Solexa Sequencer, respectively. A total 341 miRNAs in miRbase were identified and 23 potential novel miRNAs were discovered. Fisher's exact test was used to identify differentially expressed microRNAs at the 5% false discovery rate. There were 91 miRNAs differentially expressed between infected and non-infected lungs. Potential miR-146a target genes were validated by Renilla Luciferase assay. Analysis using chicken 44K Agilent microarray indicated that 146 mRNAs were differentially expressed between infected and non-infected chicken lungs. More genes (1000 vs. 303) were down-regulated with AIV infection. A comprehensive analysis combining both microRNA and targeted mRNA gene expression was developed. Results from the current study will help elucidate the lung's response to AIV. More miRNAs were most highly expressed in infected lungs (80) than non-infected lungs (11). Targets of differentially expressed miRNAs were predicted by miRanda. Thus, the differentially expressed microRNAs likely regulate host-AIV interaction and may lead to the development of novel anti-viral drugs or vaccines to control AIV infection in poultry. For Campylobacter jejuni study, following C. jejuni infection, there were more total host genes in the spleen responding to the infection in the resistant line A than in the susceptible line B. Functional analysis revealed genes related to defense response to bacteria were more highly expressed in the resistant line A than in the susceptible line B. Specifically, genes for lymphocyte activation, differentiation and humoral response, and Ig light and heavy chain were up-regulated in the resistant line. Interestingly, in the susceptible line, genes for regulation of erythrocyte differentiation, hemopoiesis, and RNA biosynthetic process were all down-regulated. An interaction analysis between genetic line and treatment demonstrated distinct defense mechanisms between lines: the resistant line promoted apoptosis and cytochrome c release from mitochondria whereas the susceptible line responded with a down-regulation of both functions. This was the first time that such defensive mechanisms against C. jejuni infection have been reported. The results of this study have uncovered novel molecular mechanisms of systemic host responses to C. jejuni infection in chickens that warrant further investigation.

Publications

Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, H. Zhou. 2011. Cecal transcriptome analysis of colonized and non-colonized chickens within two genetic lines that differ in cecal colonization by Campylobacter jejuni. Animal Genetics in press.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, H. Zhou. 2010. Gene expression profiling of the local cecal response of genetic chicken lines that differ in their susceptibility to Campylobacter jejuni colonization. PLoS ONE 5(7): e11827. doi:10.1371/journal.pone.0011827
Lee JY, Song JJ, Wooming A. Li XY, HJ Zhou, Bottje W. Kong BW. 2010 Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus. BMC Genomics 2010, 11:445.
Heidari M., A. Lopes, M. Huebner, S. Sharif, D. Kireev and H. Zhou. 2010. Mareks disease virus-induced immunosuppression: array analysis of chicken immune response genes expression profiling. Viral Immunology 23(3):309-19.

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

Outputs
OUTPUTS: For teaching, I have taught undergraduate course, Poultry Breeding and Genetics, core course (34 students) in the Spring. I also mentoring 2 Ph.D. students, two postdoctoral research associate, and one undergraduate student. For research, Avian influenza (AI) is a major respiratory disease of poultry that can cause catastrophic losses to the commercial poultry industry. It is imperative to develop effective control and treatment measures to prevent the spread of AI. The Mx protein has been shown to confer antiviral responses to influenza viruses in mouse. One nonsynonymous substitution (S631N) in the chicken Mx protein has been reported to be associated with resistance to avian influenza virus in vitro in chickens. In this study we examined the associations of Mx polymorphism with the antiviral response in chicken embryo and young chicks. The embryo and young chicks were generated from the cross of Mx heterozygous parents with expected segregating ratio of 1:2:1 in the progeny. A PCR-RFLP was developed to genotype the Mx gene (NN, NS and SS) from 119 embryos and 24 chickens. Thirteen day old embryonated chicken eggs were inoculated with 10 EID50 H5N9 AIV. Hemagglutination assay was used to evaluate virus replication in chicken embryos. Hemagglutinating units (HAU) in allantoic fluid were determined at 48 hours post-inoculation for all infected embryos. PARTICIPANTS: Ron Okimoto from Cobb Vantress is a collaborator for this project. Sanjay Reddy and Blanca Lupiani are collaborators from Texas A&M University. This project provided a training opportunity for one postdoc (Xianyao Li) and 3 Ph.D. students (Ying Wang, Wenko Chou, Xiao Li). TARGET AUDIENCES: Poultry scientific community, poultry industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
For AIV, Mean virus titers for the different phenotypes were 256 HAU for NN, 222.86 HAU for NS and 337.79 HAU for SS. For the in vivo study, 24 one-week old broilers were inoculated with 106 EID50 H5N3 and virus titers in lungs were evaluated at day 4 post inoculation. The mean virus titers for three genotypes were 398.11, 501.19 and 1000 TCID50/ml for NN, NS and SS, respectively. Our results indicate that there is a tendency for birds with NN genotypes to have lower virus titer than ones with SS both in ovo and in vivo, although the association between genotypes and virus titers was not significant (P > 0.05). Our results indicate that further studies including additional Mx alleles and more animals are needed. The knowledge generated by this study provides valuable information on the effect of the Mx gene on the genetic resistance to AIV in chickens and its potential application in the poultry breeding industry.

Publications

Wang Y., V. Brahmakshatriya, B. Lupiani, S. Reddy, B. Yoon, H. Zhu, P. Gunaratne, R. Chen, J. Wang, H. Zhou. 2009. Identification of differentially expressed microRNAs In chicken lung and trachea with avian influenza virus infection by Solexa Sequencer. BMC Genomics 10:512.
Chiang HI, LR. Berghman, H. Zhou. 2009. Inhibition of NF-kB 1 (NF-kBp50) by RNA interference in chicken macrophage HD11 cell line challenged with Salmonella enteritidis. Genetics and Molecular Biology 32:507-515.
Lu Y, Lopes A, S. Sharif, K. Zhu, H. Zhou, H. Yu, and J. Gong. 2009. Expression profiles of genes in Toll-like receptor-mediated signaling of broilers infected with Clostridium perfringens. Clinical and Vaccine Immunology 16:1639-1647.
Sarson, AJ. Wang Y, Kang Z, Dowd SE, Lu Y., Yu H., Han Y, Zhou H. and J. Gong. 2009. Gene expression profiling within the spleen of Clostridium perfringens-challenged Broilers fed antibiotic-medicated and non-medicated diets. BMC Genomics 10:260.
Satterfield, C. G. Song, K. Kochan, PK Riggs, R. Simmons, C. Elsik, D. Adelson, F. Bazer, H. Zhou, T. Spencer. 2009. Discovery of candidate genes in the endometrium regulating ovine blastocyst growth and conceptus elongation. Physiological Genomics 39:85-99.
Zhou H, J. Gong, J. Brisbin, H. Yu, A. Sarson, W. Si, S. Sharif, and Y. Han. 2009. Transcriptional Profiling Analysis of Host response to Clostridium perfringens Infection in Broilers. Poult. Sci. 88:1023-1032.
Wang J. G. Wu. H. Zhou. F. Wang. 2009. Emerging technologies for amino acid nutrition research in the post-genome era. Amino Acids. 37:177-186.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, V. DiRita, I. Y. Pevzner, H. Zhou. 2009. Gene expression profiling of innate immune response to Campylobacter jejuni infection in the bursa of broilers. Proc. Plant & Animal Genome XVII, San Diego, CA.
Wang Y., V. Brahmakshatriya, B. Lupiani, S. Reddy, B. Yoon, H. Zhu, P. Gunaratne, R. Chen, H. Zhou. 2009. Identification of differentially expressed microRNAs In chicken lung and trachea with avian influenza virus infection by Solexa Sequencer. Proc. Plant & Animal Genome XVII, San Diego, CA.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, I. Y. Pevzner, H. Zhou. 2009. Genome-wide gene expression profiling of chicken spleen responding to Campylobacter jejuni infection in broilers. Proc. Plant & Animal Genome XVII, San Diego, CA.
H.I. Chiang,, C. Swaggerty, M. Kogut, and H. Zhou. 2009. Gene expression profiling In Salmonella enterica serovar typhimurium stimulated heterophils using A chicken 44K Agilent microarray. Proc. Plant & Animal Genome XVII, San Diego, CA.
Hilley J., X. Li, C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. Genovese, H. He , V. J. Dirita, I. Pevzner, H. Zhou. 2009 Innate immune response to Campylobacter jejuni infection in the broiler bursa. 98th Annual Poultry Science meeting, Raleigh, NC. 2009.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. Genovese, H. He , I. Pevzner, H. Zhou. 2009. Gene expression profiling difference between resistant and susceptible broilers responding to Campylobacter jejuni infection. 98th Annual Poultry Science meeting, Raleigh, NC.
Chou W., X. Li, C. Bailey, H. Zhou. 2009. Gene Expression Profiling among Liver, Bone Marrow and Kidney in Broilers. 98th Annual Poultry Science meeting, Raleigh, NC.
Lee, J.Y., J.J. Song, H.Zhou, and B.W. Kong. Transcriptional profiling of host gene expression by the infection of laryngotracheitis virus in chicken embryo lung cells. 2009. Ann. Meeting of Am. Soc. for Virology, Vancouver, Canada.
Zhou H., S. Dowd, S. Lamont. 2009. Global transcriptional profiles reveal distinct mechanisms of innate response to Salmonella in three genetic lines of chickens. Proc. Plant & Animal Genome XVII, San Diego, CA.
Wang Y., V. Brahmakshatriya, B. Lupiani, S. Reddy, R. Okimoto, X. Li, H. Chiang, H. Zhou. 2009 Associations of Chicken Mx Polymorphism with Antiviral Responses in Avian Influenza Virus Infected Embryo and Broilers. 98th Annual Poultry Science meeting, Raleigh, NC.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: For teaching, I have taught undergraduate course, Poultry Breeding and Genetics, core course (44 students) in the Spring. I also mentoring 3 Ph.D. students, two postdoctoral research associate and one undergraduate student. For research, Campylobacter jejuni (C. jejuni) is a zoonotic pathogen that causes human diarrhea worldwide. Chickens are a natural reservoir of C. jejuni. Understanding the host response to C. jejuni infection at the molecular level will lay the foundation to control human campylobacterosis by reducing food contamination. Two distinct genetic lines, resistant (line A) and susceptible (line B) to C. jejuni colonization, were utilized to profile the host response to C. jejuni infection using an Agilent chicken 44K microarray. Day-old chickens were challenged orally with C. jejuni and spleens collected for total RNA 7 days post-challenge. Twenty infected samples with highest (a) or lowest bacterial number (b) in cecal content and twenty non-infected (c) in each line were randomly pooled into four biological replicates. The pair comparisons among these three groups within each line were analyzed. Innate immunity play an important role in the host response to pathogens. To study host response to C. jejuni infection in broilers, both wild-type (wt) and mutant (5 log reduction of C. jejuni colonization in the chicken gut compared to wt) C. jejuni were used to challenge day-one-old broilers. Total RNA were isolated from bursa at 1 and 4 hours post-challenge. Eight biological replicates were used in each group at each time point. The signal intensity of each gene was normalized using LOWESS method. A mixed model was used to identify differentially expressed genes by SAS (P < 0.001). 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
For Campylobacter microarray analysis in spleen, there were 468, 743, and 939 genes differentially expressed between groups a and c, groups a and b, and groups b and c in line A, respectively, and 201, 37, 126 genes in line B, respectively. More differentially expressed genes in spleen in line A than in line B were found. The results indicated that significantly different response to C. jejuni infection occurred between resistant and susceptible chicken lines, and the effects of interaction between genetics and tissue should be considered. For Campylobacter innate immune response microarray analysis, there were 908 and 583 significantly expressed genes between mutant C. jejuni infected and non-infected chickens, and between wt C. jejuni infected and non-infected chickens at 1 hour post-challenge, respectively, and 453 and 577 genes at 4 hours post-challenge, respectively. More up-regulated genes than down-regulated genes were found at 1 hour post-challenge in the comparisons of both mutant and wt vs. non-infected controls, while more down-regulated genes than up-regulated genes were detected at 4 hour post-challenge. There was a strong early host response to the mutant strain in the bursa. The results suggest there is a significantly different response caused by different C. jejuni strains within and between time points. These data provide new insights to determine the molecular mechanisms of host immunity to C. jejuni infection in broilers.

Publications

Chiang H., C. Swaggerty, M. Kogut, S. Dowd, X. Li, I. Pevzner, and H. Zhou. 2008. Gene expression profiling in chicken heterophils with Salmonella enteritidis stimulation using a chicken 44K Agilent microarray. BMC Genomics 9:526.
Li X., H.I. Chiang, J. Zhu, S. Dowd and H. Zhou. 2008. Characterization of newly developed chicken 44K Agilent microarray. BMC Genomics 9:60.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, N. J. Stern, I. Y. Pevzner, H. Zhou. 2008. The Paternal Effect of Campylobacter jejuni Colonization in Ceca in Broilers. Poult. Sci. 87:1742-1747.
Li X., C. Swaggerty, M. Kogut, H.I. Chiang, Y. Wang, K. Genovese, H. He, and H. Zhou. 2008. Global gene expression profile of chicken cecal tonsil in response to Campylobacter jejuni challenge in broiler lines. Proc. Plant & Animal Genome XVI, San Diego, CA.
Wang Y., J. Gong, Y. Han, X. Li, H.I. Chiang, Z. Kang, H. Yu, and H. Zhou. 2008. Gene expression profiling of Clostridium Perfringens infection in broilers on medicated and non-medicated diets using a chicken 44K Agilent microarray. Proc. Plant & Animal Genome XVI, San Diego, CA.
Chiang H., C. Swaggerty, M. Kogut, X. Li, and H. Zhou. 2008. Expression profiling of genes associated with Toll-like receptors signaling pathway in heterophils from Salmonella-resistant and susceptible chicken lines. Proc. Plant & Animal Genome XVI, San Diego, CA.
Li X., C. L. Swaggerty, M. H. Kogut, H. Chiang, Y. Wang, K. J. Genovese, H. He, N. J. Stern, I. Y. Pevzner, H. Zhou. 2008. The Paternal Effect of Campylobacter jejuni Colonization in Ceca in Broilers. Poult. Sci. 88: suppl. 1:68.