Damarius S. Fleming

RNA-seq analysis of broiler liver transcriptome reveals novel responses to high ambient temperature

BackgroundIn broilers, high ambient temperature can result in reduced feed consumption, digestive inefficiency, impaired metabolism, and even death. The broiler sector of the U.S. poultry industry incurs approximately

Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus

52 million in heat-related losses annually. The objective of this study is to characterize the effects of cyclic high ambient temperature on the transcriptome of a metabolically active organ, the liver. This study provides novel insight into the effects of high ambient temperature on metabolism in broilers, because it is the first reported RNA-seq study to characterize the effect of heat on the transcriptome of a metabolic-related tissue. This information provides a platform for future investigations to further elucidate physiologic responses to high ambient temperature and seek methods to ameliorate the negative impacts of heat.ResultsTranscriptome sequencing of the livers of 8 broiler males using Illumina HiSeq 2000 technology resulted in 138 million, 100-base pair single end reads, yielding a total of 13.8 gigabases of sequence. Forty genes were differentially expressed at a significance level of P-value < 0.05 and a fold-change ≥ 2 in response to a week of cyclic high ambient temperature with 27 down-regulated and 13 up-regulated genes. Two gene networks were created from the function-based Ingenuity Pathway Analysis (IPA) of the differentially expressed genes: “Cell Signaling” and “Endocrine System Development and Function”. The gene expression differences in the liver transcriptome of the heat-exposed broilers reflected physiological responses to decrease internal temperature, reduce hyperthermia-induced apoptosis, and promote tissue repair. Additionally, the differential gene expression revealed a physiological response to regulate the perturbed cellular calcium levels that can result from high ambient temperature exposure.ConclusionsExposure to cyclic high ambient temperature results in changes at the metabolic, physiologic, and cellular level that can be characterized through RNA-seq analysis of the liver transcriptome of broilers. The findings highlight specific physiologic mechanisms by which broilers reduce the effects of exposure to high ambient temperature. This information provides a foundation for future investigations into the gene networks involved in the broiler stress response and for development of strategies to ameliorate the negative impacts of heat on animal production and welfare.

Impacts of Salmonella enteritidis infection on liver transcriptome in broilers

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library.ResultsMajor changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold.ConclusionsThe magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

Single nucleotide variant discovery of highly inbred Leghorn and Fayoumi chicken breeds using pooled whole genome resequencing data reveals insights into phenotype differences

Salmonella enterica serovar enteritidis is an enteric bacterium that can contaminate chicken eggs and meat, resulting in production losses and consumer illness. To provide insight into the systemic metabolic effects of S. enteritidis infection, liver samples were harvested 10‐days postinfection from broiler hens. Hepatic global gene expression levels were assessed using a chicken 44K Agilent microarray. Forty‐four genes were differentially expressed at a significance level of q value < 0.05. One hundred eighty‐three genes were differentially expressed at a suggestive significance level of q value < 0.1. A predominance of downregulation existed among significantly differentially expressed genes. Cell cycle and metabolism networks were created from the differentially expressed genes. Mitochondria‐mediated apoptosis, electron transport, peptidase activity, vein constriction, cell differentiation, IL‐2 signaling, Jak‐Stat signaling, B‐cell receptor signaling, GDP/GTP exchange, and protein recycling were among the functions of the differentially expressed genes that were down‐regulated in response to S. enteritidis. The effects of S. enteritidis infection on the liver transcriptome profiles of broilers reflect a predominance of downregulation of genes with cell cycle and metabolic functions. The most pronounced response was the downregulation of genes that function in metabolic pathways, inflammation, and mitochondria‐mediated apoptosis. These results provide insight into important systemic metabolic mechanisms that are active in the chicken liver in response to S. enteritidis infection at 10‐days postinfection. genesis 51:357–364, 2012.

Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived cells to different stimuli.

BackgroundAnalyses of sequence variants of two distinct and highly inbred chicken lines allowed characterization of genomic variation that may be associated with phenotypic differences between breeds. These lines were the Leghorn, the major contributing breed to commercial white-egg production lines, and the Fayoumi, representative of an outbred indigenous and robust breed. Unique within- and between-line genetic diversity was used to define the genetic differences of the two breeds through the use of variant discovery and functional annotation.ResultsDownstream fixation test (FST) analysis and subsequent gene ontology (GO) enrichment analysis elucidated major differences between the two lines. The genes with high FST values for both breeds were used to identify enriched gene ontology terms. Over-enriched GO annotations were uncovered for functions indicative of breed-related traits of pathogen resistance and reproductive ability for Fayoumi and Leghorn, respectively.ConclusionsVariant analysis elucidated GO functions indicative of breed-predominant phenotypes related to genomic variation in the lines, showing a possible link between the genetic variants and breed traits.

Genomic Comparison of Indigenous African and Northern European Chickens Reveals Putative Mechanisms of Stress Tolerance Related to Environmental Selection Pressure

Monocyte-derived DCs (mDCs) are major target cells in porcine reproductive and respiratory syndrome virus (PRRSV) pathogenesis; however, the plasticity of mDCs in response to activation stimuli and PRRSV infection remains unstudied. In this study, we polarized mDCs, and applied genome-wide transcriptomic analysis and predicted protein-protein interaction networks to compare signature genes involved in mDCs activation and response to PRRSV infection. Porcine mDCs were polarized with mediators for 30 hours, then mock-infected, infected with PRRSV strain VR2332, or a highly pathogenic PRRSV strain (rJXwn06), for 5 h. Total RNA was extracted and used to construct sequencing libraries for RNA-Seq. Comparisons were made between each polarized and unpolarized group (i.e. mediator vs. PBS), and between PRRSV-infected and uninfected cells stimulated with the same mediator. Differentially expressed genes (DEG) from the comparisons were used for prediction of interaction networks affected by the viruses and mediators. The results showed that PRRSV infection inhibited M1-prone immune activity, downregulated genes, predicted network interactions related to cellular integrity, and inflammatory signaling in favor of M2 activity. Additionally, the number of DEG and predicted network interactions stimulated in HP-PRRSV infected mDCs was superior to the VR-2332 infected mDCs and conformed with HP-PRRSV pathogenicity.

Genomic analysis of Ugandan and Rwandan chicken ecotypes using a 600 k genotyping array

Global climate change is increasing the magnitude of environmental stressors, such as temperature, pathogens, and drought, that limit the survivability and sustainability of livestock production. Poultry production and its expansion is dependent upon robust animals that are able to cope with stressors in multiple environments. Understanding the genetic strategies that indigenous, noncommercial breeds have evolved to survive in their environment could help to elucidate molecular mechanisms underlying biological traits of environmental adaptation. We examined poultry from diverse breeds and climates of Africa and Northern Europe for selection signatures that have allowed them to adapt to their indigenous environments. Selection signatures were studied using a combination of population genomic methods that employed FST, integrated haplotype score (iHS), and runs of homozygosity (ROH) procedures. All the analyses indicated differences in environment as a driver of selective pressure in both groups of populations. The analyses revealed unique differences in the genomic regions under selection pressure from the environment for each population. The African chickens showed stronger selection toward stress signaling and angiogenesis, while the Northern European chickens showed more selection pressure toward processes related to energy homeostasis. The results suggest that chromosomes 2 and 27 are the most diverged between populations and the most selected upon within the African (chromosome 27) and Northern European (chromosome 2) birds. Examination of the divergent populations has provided new insight into genes under possible selection related to tolerance of a population’s indigenous environment that may be baselines for examining the genomic contribution to tolerance adaptions.