Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: Liang Sun (graduate student) under the direction of Carl Schmidt (Avian Biosciences Center Faculty) has been investigating heat stress response in chickens. In particular, he has been preparing transcriptome libraries to describe gene expression patterns in liver, and liver cell lines, in response to heat. 40 different libraries have been constructed from samples raised at normal or stressed temperatures. These libraries provide gene expression levels for all 15,495 genes currently recognized in the chicken genome. While data analysis is ongoing, these experiments have identified both previously recognized and novel genes that are either induced or repressed by heat. The long-term goal is to integrate these studies with quantitative trait studies to improve the chicken's ability to tolerate heat. Christopher Resnyk is a Master of Science (MSc) degree candidate in the Department of Animal and Food Sciences (ANFS) under the direction of Larry Cogburn (Avian Biosciences Center Faculty). Chris' Master's research project is focused on involvement of adipokines and retinoic acid signaling in lipogenesis and development of adiposity in the chicken. During the spring semester and summer, Chris worked on our microarray analysis of the adipose transcriptome in genetically fat (FL) and lean (LL) chickens. He also verified differential expression of dozens of candidate genes using quantitative real-time PCR (qRT-PCR) analysis. This transcriptional analysis of adipose tissue has revealed higher expression of a large number of genes involved in the blood coagulation cascade in the genetically lean chickens when compared to the fat chickens. A large number of retinoid-induced genes are also highly expressed in adipose tissue of lean chickens. These findings were originally presented in the American Physiological Society poster session on Physiological Genomics of Exercise, Metabolism, and Obesity at the Experimental Biology Conference (EB-2011) in Washington, DC, April 9-13, 2011. Chris has also recently completed first draft of a paper on microarray analysis of abdominal fatness in the FL and LL chickens which to be submitted to BMC Genomics. PARTICIPANTS: Jack Gelb, Ph.D Carl Schmidt, Ph.D Larry Cogburn, Ph.D Mark Parcells, Ph.D Liang Sun, Graduate Student Upendra Katneni, Graduate Student Christopher Resnyk, Graduate Student TARGET AUDIENCES: Amican Physiological Society; International Symposium on Marek's Disease, Berlin, 2012; Northeastern Conference on Avian Diseases; Plant and Animal Genome Meeting PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The focus of Upendra Katneni's (graduate student) work under the direction of Mark Parcells (Avian Biosciences Center Faculty) has been on understanding the immune signature elicited by vaccines. He has contributed to several projects including the cloning and expression of several genes of ILTV, the characterization of recombinant Marek's diseases viruses, as well as the development of reporters for type I interferon and pro-inflammatory cytokine genes. His current and future project is focused on generating reporter cell lines for assessing innate immune signaling. These will be used to assess the integrated effects of vaccination for Marek's disease, as well as the effects of innate immune inducers. These will provide invaluable insight into innate immune signaling during vaccination. He also performs Analysis of the abdominal fat transcriptome uncovered differential expression of a large number of genes that regulate metabolism, hemostasis, retinoid signaling, and adipogenesis. The excessive deposition of abdominal fat in the FL appears to result from dysregulation of signaling between major endocrine organs, especially the liver (primary site of lipogenesis) and adipose tissue (which exerts its action through the release of adipokines). For his Master's degree program, Chris Resynk continues to receive cross-discipline training in molecular biology, genetics, bioinformatics and computational biology. Chris is applying these computational tools to better understand the role of retinoid-induced adipokines in development of obesity and type-2 diabetes, using the chicken as a biomedical model. Chris and Dr. Cogburn have recently completed RNA-seq analysis of 8 bar-coded cDNA libraries constructed from RNA isolated from abdominal fat of 4 fat (FL) and 4 lean (LL) line chickens at 7 wk of age (Cogburn et al., unpublished observations). The 8 libraries were paired-end (2 x 101 bp) sequenced on an Illumina HiSeq 2000 sequencer in two configurations: a single pool of all eight libraries sequenced in a single lane or two pools (each made from 2 FL and 2 LL birds) sequenced in two lanes. The total number of paired-end (2 x 101 bp) reads achieved in this preliminary RNA-seq study was 59 million (M) for the LL and 51M for the FL abdominal fat libraries. Our RNA-seq analysis has identified 1704 differentially expressed (DE) genes in adipose tissue of FL and LL chickens, which exhibit a 3-fold difference in visceral fatness at this age. Chris has confirmed the differential expression of dozens of candidate genes in abdominal fat of FL and LL chickens using qRT-PCR analysis. Remarkably, adipose tissue of LL chickens shows higher expression of a large number of genes involved in blood clotting (hemostasis), retinoid signaling and lipolysis. RNAseq analysis has revealed the "ectopic" expression of additional regulators of lipogenesis, adiposity and lipolysis. Surprisingly, adipose tissue produces the pancreatic hormone glucagon (GCG) and hypothalamic regulators of energy intake and metabolism [cannabinoid receptor (CNR1), fatty acid amide hydrolase (FAAH) and neuropeptide Y (NPY)].
Publications
- Resnyk, C., C. Chen, H. Huang, C.H. Wu, E. Le Bihan-Duval, M. Duclos, J. Simon and L. A. Cogburn, 2012. Systems biology of adiposity in genetically fat and lean chickens: Unique biomedical models of visceral obesity. CBCB Research Symposium on Bioinformatics and Systems Biology, University of Delaware. May 24, 2012.
- Resnyk, C.W., W. Carre, X. Wang, E. Le Bihan-Duval, M. J. Duclos, B. Leclercq, J. Simon, S.E. Aggrey, T. E. Porter, and L. A. Cogburn, 2012. Transcriptional analysis of abdominal fat accretion in genetically fat and lean chickens reveals link between hemostatic genes and leanness. BMC Genomics (for submission on September 15, 2012)
- Resnyk, C., C. Chen, H. Huang, C.H. Wu, E. Le Bihan-Duval, M. Duclos, J. Simon and L. A. Cogburn, 2012. RNA-Seq analysis of abdominal fat in genetically fat and lean chickens reveals ectopic expression of hemostatic and endocrine factors controlling metabolism. BMC Genomics (for submission on October 30, 2012)
- Shaikh, S. L., U. Katneni, H. Dong, S. Gaddamanugu, P. Tavlarides-Hontz, K. Jarosinski, N. Osterrieder, and M.S. Parcells. 2012. A naturally-occurring mutation in the glycoprotein L (gL) gene of MDV field strains in the US does not confer increased pathogenicity to the BAC-based RB-1B strain. 9th International Symposium on Marek's Disease (Berlin, June 24 - 28)
- Kumar, P. M., H. Dong, D. Lenihan, S. Gaddamanugu, U. Katneni, S. Shaikh, P. Tavlarides-Hontz, S. Reddy, and M. S. Parcells. 2012. Selection of a recombinantn Marek's disease virus (MDV) in vivo through expression of the Meq oncoprotein: characterization of an rMd5-based mutant expressing the Meq protein of strain RB-1B. Avian Disease 56: 328-340.
- Gaddamanugu, S., B. Ladman,L. Preskenis, U. K. Katneni, P. Tavlarides-Hontz, and M. S. Parcells. 2011. Cloning and expression of the UL48 (VP16) and ICP4 proteins of Infectious Laryngotracheitis virus (ILTV), Northeastern Conference on Avian Diseases.
- Katneni, U.K., E. McDowell, H. Dong, S. Shaikh, S. Gaddamanugu, P. Tavlarides-Hontz, and M. S. Parcells. 2011. Mareks disease virus (MDV)-based vaccines lacking the Meq oncoprotein, Northeastern Conference on Avian Diseases.
- Rojas J., E. McDowell, U. Katneni, K. Jarosinski, and M. S. Parcells. 2011. Targeting Mareks disease virus (MDV) transmission via vaccination, Northeastern Conference on Avian Diseases.
- Cogburn, LA, Resnyk, C, TE Porter, SE Aggrey, E Le Bihan-Duval, M Duclos, and J Simon, 2011. Transcriptional analysis of abdominal fat accretion in genetically fat and lean chickens: A new polygenic model of visceral obesity FASEB J (March 17, 2011) 25:862.7.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Campylobacter jejuni prevalence in agriculturally associated and migratory wild birds. Judith I. Keller (Graduate Student) & W. Gregory Shriver (Avian Biosciences Center Faculty). Campylobacter bacteria negatively impact human health, being responsible for the majority of food borne illnesses and gastroenteritis. Understanding the prevalence of bacterial pathogens in environmental factors influencing the food industry, such as wild birds, may be used to examine the spread of the pathogens. Wild birds are thought to contribute to the spread of Campylobacter, but few studies have estimated the prevalence of Campylobacter in North American species (but see Keller et al. 2011). The objectives of this study were; 1) determine the prevalence of C. jejuni, C. coli and C. lari in agriculturally associated wild birds and 2) determine the presence of wild-bird related strains of C. jejuni in backyard poultry. Samples were plated on selective agar (CAMP) for 42C for 48h under microaerobic conditions. If growth occured, samples were replated on CAMP and isolated colonies were grown microaerobically (CO2) and in O2 rich environment at 42C for 48h. If no growth occurred on O2 CAMP, samples were tested for positive catalase and oxidase reactions and plated on Sheep Blood Agar. If characteristic growth occurred, cell morphology was determined and samples were prepared for DNA extraction and multiplex (MX) and multi-locus sequence typing (MLST)PCR. Role of microRNAs in Salmonella infections in the chicken. Robin Morgan (ABC Faculty), Erin Bernberg (ABC Scientist) and Natalie Stevenson(Undergraduate Student). MicroRNAs (miR) are short PNA sequences, approx. 22nt in length, which play a role in post-transcriptional regulation of gene expression. One important miR is miR-155, which is involved in the differentiation of lymphocytes. Viral versions of this miR function in the pathogenesis of Marek's disease (MD) in poultry, a herpesvirus-induced T-cell lymphoma in chickens, and Epstein-Barr virus infections in humans. A miR from MD virus (MDV), MDV1-miR-M4, shares a seed sequence with miR-155. Our laboratory inserted MDV1-miR-M4 into the herpesvirus of turkeys (HVT) genome to generate an HVT-M4 construct. HVT is currently used as a vaccine against the tumor formation in MDV-infected chickens. Preliminary results indicate that HVT-M4 results in higher loads of re-isolatable virus post inoculation compared to the HVT parent virus. Expression of MDV1-miR-M4 may increase lymphocyte differentiation resulting in more lymphocytes to propagate HVT, a scenario that could augment immune responsiveness to MDV and perhaps other pathogens. In contrast to MDV, little is known about the role of miRs in Salmonella infections of chickens. Salmonella are a source of human food-borne infections and cause significant health costs. It is possible that miR-155 plays a role in the response to Salmonella infection in chickens. It has already been shown that mice lacking miR-155 fail to respond normally to Salmonella vaccination. If the same holds true for chickens, it stands to reason that vaccination of chickens with the HVT-M4 may increase resistance to infection by Salmonella. 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
Campylobacter jejuni prevalence in agriculturally associated and migratory wild birds. Judith I. Keller (Graduate Student) and W. Gregory Shriver (Avian Biosciences Center Faculty). We collected 665 wild bird samples at 26 and 45 domestic chicken samples from eight sites distributed in Delaware, Pennsylvania, and New Jersey. Results from culturing methods indicated that 30% of snow geese and 6% of canada geese were positive for Campylobacter spp. Both migratory shorebird species sampled in the Delaware Bay were positive for Campylobacter spp., including C. jejuni; ruddy turnstones had a prevalence of 43%, sanderling 4%, and semipalmated sandpipers 26%. Of several Laridae species, 12% were positive for Campylobacter spp. including C. jejuni and C. lari. We detected Campylobacter spp. in 9% of the domestic backyard chickens sampled. Through multilocus sequence typing PCR specific to C. jejuni, 10% of the wild bird samples were positive for C. jejuni, 1.2% were positive for C. coli and 0.3% were positive for C. lari. Wintering waterfowl and gulls often occur on agricultural fields possibly spreading disease organisms. Shorebirds undergo migrations and have the potential of transferring bacteria and antibiotic resistant genes across long distances. With implications of wild birds transmitting the Campylobacter bacteria to other avian species, such as poultry, this study provides further insight into C. jejuni occurrence within selected wild bird populations in North America, USA, but also provides insight into the difference in infection of commercial poultry farms as compared to backyard poultry. We are currently in the process of further analyzing samples by sequencing MLST PCR products. Samples whose PCR product was positive for all 7 housekeeping gene PCR reactions were purified using a QiaQuick PCR purification system employing spin-columns (Qiagen). Samples and sequencing primers also from the pubmlst.org/campylobacter website overseen by Dingle et al. (2001) were sent to the University of Delaware Biotechnology Institute's Sequencing and Genotyping Center for sequencing. Role of microRNAs in Salmonella infections in the chicken. Robin Morgan (ABC Faculty), Erin BernBerg (Department of Animal and Food Sceinces) and Natalie Stevenson(Undergraduate Student). In order to test whether HVT-M4 increases Salmonella resistance or augments immune responses to Salmonella vaccination,experiments are planned to test the effect of inoculating eggs with rHVT-M4 compared to the HVT parent on Salmonella vaccination and challenge.
Publications
- KELLER, J. I., SHRIVER, W. G., WALDENSTROM, J., GRIEKSPOOR, P., OLSEN, B. 2011. Prevalence of Campylobacter in Wild Birds of the Mid-Atlantic Region, USA. Journal of Wildlife Diseases 47:750-754.
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