Caecal transcriptome analysis of colonized and non-colonized chickens within two genetic lines that differ in caecal colonization by Campylobacter jejuni

Campylobacter jejuni is one of the most common causes of human bacterial enteritis worldwide. The molecular mechanisms of the host responses of chickens to C. jejuni colonization are not well understood. We have previously found differences in C. jejuni colonization at 7‐days post‐inoculation (pi) between two genetic broiler lines. However, within each line, not all birds were colonized by C. jejuni (27.5% colonized in line A, and 70% in line B). Therefore, the objective of the present experiments was to further define the differences in host gene expression between colonized and non‐colonized chickens within each genetic line. RNA isolated from ceca of colonized and non‐colonized birds within each line was applied to a chicken 44K Agilent microarray for the pair comparison. There were differences in the mechanisms of host resistant to C. jejuni colonization between line A and line B. Ten times more differentially expressed genes were observed between colonized and non‐colonized chickens within line B than those within line A. Our study supports the fact that the MAPK pathway is important in host response to C. jejuni colonization in line B, but not in line A. The data indicate that inhibition of small GTPase‐mediated signal transduction could enhance the resistance of chickens to C. jejuni colonization and that the tumour necrosis factor receptor superfamily genes play important roles in determining C. jejuni non‐colonization in broilers.     

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

ABSTRACT: BACKGROUND: Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited. RESULTS: Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6M and 3.3M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher's exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection. CONCLUSION: A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122-1, 122-2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken.     

Splenic gene expression profiling in White Leghorn layer inoculated with the Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (SE) is a foodborne pathogen that can threaten human health through contaminated poultry products. Live poultry, chicken eggs and meat are primary sources of human salmonellosis. To understand the genetic resistance of egg‐type chickens in response to SE inoculation, global gene expression in the spleen of 20‐week‐old White Leghorn was measured using the Agilent 4 × 44 K chicken microarray at 7 and 14 days following SE inoculation (dpi). Results showed that there were 1363 genes significantly differentially expressed between inoculated and non‐inoculated groups at 7 dpi (I7/N7), of which 682 were up‐regulated and 681 were down‐regulated genes. By contrast, 688 differentially expressed genes were observed at 14 dpi (I14/N14), of which 371 were up‐regulated genes and 317 were down‐regulated genes. There were 33 and 28 immune‐related genes significantly differentially expressed in the comparisons of I7/N7 and I14/N14 respectively. Functional annotation revealed that several Gene Ontology (GO) terms related to immunity were significantly enriched between the inoculated and non‐inoculated groups at 14 dpi but not at 7 dpi, despite a similar number of immune‐related genes identified between I7/N7 and I14/N14. The immune response to SE inoculation changes with different time points following SE inoculation. The complicated interaction between the immune system and metabolism contributes to the immune responses to SE inoculation of egg‐type chickens at 14 dpi at the onset of lay. GC, TNFSF8, CD86, CD274, BLB1 and BLB2 play important roles in response to SE inoculation. The results from this study will deepen the current understanding of the genetic response of the egg‐type chicken to SE inoculation at the onset of egg laying.     

利用基因芯片技术研究两品种鸡脂肪组织差异表达基因

应用包含9024条鸡cDNA的表达谱芯片,对从两品种鸡脂肪组织抽提及纯化的mRNA进行芯片杂交,并对基因表达谱进行分析,旨在筛选高脂肉鸡和白耳蛋鸡脂肪组织差异表达的基因,探讨造成两品种体脂性状差异的分子生物学机理。结果按差异显著阳性标准分析,共筛选出差异表达基因67条,主要涉及脂类代谢、能量代谢、细胞骨架构成、转录和剪接因子以及蛋白质合成与分解等相关基因,此外,还筛选出一些尚未在GenBank上登陆的序列,推测可能是未知的新基因,它们在鸡脂类代谢的过程所起到的作用还需进一步实验证明。     

Candidate gene approach: potentional association of caspase-1, inhibitor of apoptosis protein-1, and prosaposin gene polymorphisms with response to Salmonella enteritidis challenge or vaccination in young chicks

Salmonella enteritidis (SE) contamination of poultry products is a major cause of foodborne disease worldwide. Caspase-1 and inhibitor of apoptosis protein-1 (IAP-1) were selected as candidate genes for chicken response to SE because their proteins play critical roles in the apoptotic pathway when intracellular bacteria interact with host cells. Prosaposin (PSAP) was selected as a positional candidate gene based on a previous quantitative trait loci (QTL) linkage study using the same population. The F1 offspring of outbred sires crossed with three diverse, highly inbred dam lines (two major histocompatibility complex-congenic Leghorn lines named G-B1 and G-B2, and one Fayoumi line) were used to define the phenotypes. The F1 birds were involved in either pathogenic SE challenge, in which spleen and cecum content bacterial load were quantified, or SE vaccination, in which plasma antibody level to SE vaccine was evaluated. A polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay was developed to identify single-nucleotide polymorphism (SNP) in the three genes. The F1 offspring of heterozygous sires for each gene were genotyped. The sire caspase-1 gene was significantly associated with cecum content bacterial load (P = 0.04) in the three combined dam line crosses, and with spleen bacterial load in the G-B1 cross (P=0.02). The sire caspase-1 gene was also significantly associated with antibody level to SE vaccine (P=0.03) in F1 males in the three combined dam line crosses. The sire IAP-1 gene was significantly associated with spleen bacterial load (P=0.04) in the three combined dam-line crosses, and interacted with dam-line genetics (P = 0.01) for cecum content bacterial load. The sire PSAP gene significantly interacted with sex for spleen bacterial load (P = 0.004). This study is the first to demonstrate the association of SNPs for caspase-1, IAP-1, and PSAP genes with SE vaccine and with pathogen challenge response in chickens.     

RNA-Seq Analysis of Abdominal Fat Reveals Differences between Modern Commercial Broiler Chickens with High and Low Feed Efficiencies

For economic and environmental reasons, chickens with superior feed efficiency (FE) are preferred in the broiler chicken industry. High FE (HFE) chickens typically have reduced abdominal fat, the major adipose tissue in chickens. In addition to its function of energy storage, adipose tissue is a metabolically active organ that also possesses endocrine and immune regulatory functions. It plays a central role in maintaining energy homeostasis. Comprehensive understanding of the gene expression in the adipose tissue and the biological basis of FE are of significance to optimize selection and breeding strategies. Through gene expression profiling of abdominal fat from high and low FE (LFE) commercial broiler chickens, the present study aimed to characterize the differences of gene expression between HFE and LFE chickens. mRNA-seq analysis was carried out on the total RNA of abdominal fat from 10 HFE and 12 LFE commercial broiler chickens, and 1.48 billion of 75-base sequence reads were generated in total. On average, 11,565 genes were expressed (>5 reads/gene/sample) in the abdominal fat tissue, of which 286 genes were differentially expressed (DE) at q (False Discover Rate) < 0.05 and fold change > 1.3 between HFE and LFE chickens. Expression levels from RNA-seq were confirmed with the NanoString nCounter analysis system. Functional analysis showed that the DE genes were significantly (p < 0.01) enriched in lipid metabolism, coagulation, and immune regulation pathways. Specifically, the LFE chickens had higher expression of lipid synthesis genes and lower expression of triglyceride hydrolysis and cholesterol transport genes. In conclusion, our study reveals the overall differences of gene expression in the abdominal fat from HFE and LFE chickens, and the results suggest that the divergent expression of lipid metabolism genes represents the major differences.     

Comparative RNA-Seq analysis reveals insights in Salmonella disease resistance of chicken; and database development as resource for gene expression in poultry

Salmonella, one of the major infectious diseases in poultry, causes considerable economic losses in terms of mortality and morbidity, especially in countries that lack effective vaccination programs. Besides being resistant to diseases, indigenous chicken breeds are also a potential source of animal protein in developing countries. For understanding the disease resistance, an indigenous chicken line Kashmir faverolla, and commercial broiler were selected. RNA-seq was performed after challenging the chicken with Salmonella Typhimurium. Comparative differential expression results showed that following infection, a total of 3153 genes and 1787 genes were differentially expressed in the liver and spleen, respectively. The genes that were differentially expressed included interleukins, cytokines, NOS2, Avβ-defensins, toll-like receptors, and other immune-related gene families. Most of the genes and signaling pathways involved in the innate and adaptive immune responses against bacterial infection were significantly enriched in the Kashmir faverolla. Pathway analysis revealed that most of the enriched pathways were MAPK signaling pathway, NOD-like receptor signaling pathway, TLR signaling pathway, PPAR signaling pathway, endocytosis, etc. Surprisingly some immune-related genes like TLRs were upregulated in the susceptible chicken breed. On postmortem examination, the resistant birds showed small lesions in the liver compared to large necrotic lesions in susceptible birds. The pathological manifestations and RNA sequencing results suggest a balancing link between resistance and infection tolerance in Kashmir faverolla. Here we also developed an online Poultry Infection Database (https://skuastk.org/pif/index.html), the first publicly available gene expression resource for disease resistance in chickens. The available database not only shows the data for gene expression in chicken tissues but also provides quick search, visualization and download capacity.     

Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation,using expressed sequence tags (ESTs) analysis and cDNA microarrays

To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified.The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.     

Dietary protein regulates in vitro lipogenesis and lipogenic gene expression in broilers

The purpose of this experiment was to determine the possible relationship between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 21 or 30% protein ad libitum. In addition, another group of birds was fed on a regimen consisting of a daily change in the dietary protein level (12 or 30%). This latter group was further subdivided such that one-half of the birds received each level of protein on alternating days. Birds were sampled from 28 to 30 days of age. Measurements taken included in vitro lipogenesis, malic enzyme activity the expression of the genes for malic enzyme, fatty acid synthase and acetyl coenzyme carboxylase. In vitro lipogenesis and malic enzyme activity were inversely related to dietary protein levels (12-30%) and to acute changes from 12 to 30%. In contrast, expression of malic enzyme, fatty acid synthase and acetyl CoA carboxylase genes were constant over a dietary protein range of 12-21%, but decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out, however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.     

The optimal discovery procedure for large-scale significance testing, with applications to comparative microarray experiments

As much of the focus of genetics and molecular biology has shifted toward the systems level, it has become increasingly important to accurately extract biologically relevant signal from thousands of related measurements. The common property among these high-dimensional biological studies is that the measured features have a rich and largely unknown underlying structure. One example of much recent interest is identifying differentially expressed genes in comparative microarray experiments. We propose a new approach aimed at optimally performing many hypothesis tests in a high-dimensional study. This approach estimates the optimal discovery procedure (ODP), which has recently been introduced and theoretically shown to optimally perform multiple significance tests. Whereas existing procedures essentially use data from only one feature at a time, the ODP approach uses the relevant information from the entire data set when testing each feature. In particular, we propose a generally applicable estimate of the ODP for identifying differentially expressed genes in microarray experiments. This microarray method consistently shows favorable performance over five highly used existing methods. For example, in testing for differential expression between two breast cancer tumor types, the ODP provides increases from 72% to 185% in the number of genes called significant at a false discovery rate of 3%. Our proposed microarray method is freely available to academic users in the open-source, point-and-click EDGE software package.     

致心律失常型右室心肌病引起的心力衰竭分子标志物的筛选

目的：筛选致心律失常型右室心肌病引起心力衰竭的分子标志物。方法：从本院的心脏病组织库中挑选5例病理诊断明确和各方面资料比较齐全的致心律失常型右室心肌病引起心力衰竭的心脏病标本(来源于心脏移植的受体),与年龄、性别和种族等因素相匹配的正常对照心脏组织(来源于心脏移植的供体)进行全基因组表达芯片的比较研究。提取致心律失常型右室心肌病的左心室组织RNA,同时提取正常对照心脏相应部位的RNA。应用晶芯人类全基因组寡核苷酸微阵列基因表达谱芯片(含有人类基因35,000个),筛选致心律失常型右室心肌病引起的心力衰竭基因表达谱的改变。应用实时定量荧光反转录聚合酶链式反应(real-time RT-PCR)验证致心律失常型右室心肌病引起的心力衰竭基因表达改变的真实性和准确性。 结果：应用基因表达芯片研究方法共筛选出78个差异表达基因,其中有35个基因在致心律失常型右室心肌病引起的心力衰竭中表达升高,而另有43个基因表达降低。其中变化较多的基因属于与代谢相关的基因。对其中36个差异表达基因应用real-time RT-PCR的方法进行了验证,差异表达基因的准确性在75%,并首次报告了心钠素在致心律失常型右室心肌病引起的心力衰竭中表达明显升高。结论：本研究在世界上首次应用基因表达芯片的方法,观察了致心律失常型右室心肌病引起的心力衰竭基因表达谱的改变,为致心律失常型右室心肌病引起的心力衰竭分子机制的阐明和寻找疾病特异的分子标志物,以用于鉴别诊断、判断病情和预后及指导个性化治疗奠定了基础。     

Identification of germinal disk region derived genes potentially involved in hen fertility

Despite the regular decrease in fertility observed in hens, especially in “meat” lines, little is known about genes affecting fertility. We used the Affymetrix microarray to search for oocyte genes whose expression would vary in relation to fertility rate in both “laying” and “meat” line hens. We focused on oocyte genes because several of them have been found to be involved in fertility in other species. Based on microarray analysis, 54 and 84 genes were differentially expressed between germinal disc regions (GDR) of F1 maturation stage oocytes from hens exhibiting either high (100%) or low (from 22% to 80%) fertility rate from laying and meat lines respectively. Most of these differentially expressed genes were distributed between “laying” and “meat” lines indicating that mechanisms involved in the decrease in fertility rates in these two cases were independent. Real time RT‐PCR performed on the same samples which were used for microarray confirmed in several cases differences in gene expression levels detected by microarray. Moreover the correlations between gene expression levels and fertility rates were evaluated for the 10 most interesting genes at different stages of follicular maturation and early embryo development on individual GDR samples from hens exhibiting different fertility rates. In total, we identified five genes whose expression levels correlated with fertility rate in accordance with findings of microarray analysis and real time RT‐PCR: VWC2, CR407412, TAPA, FGL2, and TRAP6. The biological significance of these genes sheds light on potential mechanisms influencing fertility and could provide candidates for fertility markers in the hen. Mol. Reprod. Dev. 76: 1043–1055, 2009. © 2009 Wiley‐Liss, Inc.