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Deep sequencing-based transcriptome analysis of chicken spleen in response to avian pathogenic Escherichia coli (APEC) infection.

ABSTRACT: Avian pathogenic Escherichia coli (APEC) leads to economic losses in poultry production and is also a threat to human health. The goal of this study was to characterize the chicken spleen transcriptome and to identify candidate genes for response and resistance to APEC infection using Solexa sequencing. We obtained 14422935, 14104324, and 14954692 Solexa read pairs for non-challenged (NC), challenged-mild pathology (MD), and challenged-severe pathology (SV), respectively. A total of 148197 contigs and 98461 unigenes were assembled, of which 134949 contigs and 91890 unigenes match the chicken genome. In total, 12272 annotated unigenes take part in biological processes (11664), cellular components (11927), and molecular functions (11963). Summing three specific contrasts, 13650 significantly differentially expressed unigenes were found in NC Vs. MD (6844), NC Vs. SV (7764), and MD Vs. SV (2320). Some unigenes (e.g. CD148, CD45 and LCK) were involved in crucial pathways, such as the T cell receptor (TCR) signaling pathway and microbial metabolism in diverse environments. This study facilitates understanding of the genetic architecture of the chicken spleen transcriptome, and has identified candidate genes for host response to APEC infection.

SUBMITTER: Nie Q

PROVIDER: S-EPMC3409229 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Deep sequencing-based transcriptome analysis of chicken spleen in response to avian pathogenic Escherichia coli (APEC) infection.

Nie Qinghua Q Sandford Erin E EE Zhang Xiquan X Nolan Lisa K LK Lamont Susan J SJ

PloS one 20120731 7

Avian pathogenic Escherichia coli (APEC) leads to economic losses in poultry production and is also a threat to human health. The goal of this study was to characterize the chicken spleen transcriptome and to identify candidate genes for response and resistance to APEC infection using Solexa sequencing. We obtained 14422935, 14104324, and 14954692 Solexa read pairs for non-challenged (NC), challenged-mild pathology (MD), and challenged-severe pathology (SV), respectively. A total of 148197 conti ...[more]

PMID: 22860004

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Project description:BackgroundAvian pathogenic Escherichia coli (APEC) is a major cause of disease impacting animal health. The bone marrow is the reservoir of immature immune cells; however, it has not been examined to date for gene expression related to developmental changes (cell differentiation, maturation, programming) after APEC infection. Here, we study gene expression in the bone marrow between infected and non-infected animals, and between infected animals with mild (resistant) versus severe (susceptible) pathology, at two times post-infection.ResultsWe sequenced 24 bone marrow RNA libraries generated from the six different treatment groups with four replicates each, and obtained an average of 22 million single-end, 100-bp reads per library. Genes were detected as differentially expressed (DE) between APEC treatments (mild pathology, severe pathology, and mock-challenged) at a given time point, or DE between 1 and 5 days post-infection (dpi) within the same treatment group. Results demonstrate that many immune cells, genes and related pathways are key contributors to the different responses to APEC infection between susceptible and resistant birds and between susceptible and non-challenged birds, at both times post-infection. In susceptible birds, lymphocyte differentiation, proliferation, and maturation were greatly impaired, while the innate and adaptive immune responses, including dendritic cells, monocytes and killer cell activity, TLR- and NOD-like receptor signaling, as well as T helper cells and many cytokine activities, were markedly enhanced. The resistant birds' immune system, however, was similar to that of non-challenged birds.ConclusionThe DE genes in the immune cells and identified signaling models are representative of activation and resolution of infection in susceptible birds at both post-infection days. These novel results characterizing transcriptomic response to APEC infection reveal that there is combinatorial activity of multiple genes controlling myeloid cells, and B and T cell lymphopoiesis, as well as immune responses occurring in the bone marrow in these early stages of response to infection.

Project description:Purpose: This study is to more comprehensively understand the genome-wide host response to avian pathogen E. coli (APEC). Methods: Male broiler chicks were challenged with APEC (or mock-challenged as controls), and bone marrow was harvested at 1 and 5 days post-infection (dpi). Based upon necropsy-scored lesions on liver, pericardium, and air sacs, the challenged birds were assigned to mild or severe pathology categories, representing resistant and susceptible phenotypes, respectively. RNA sequence data were first analyzed using the R package EdgeR to identify differentially expressed genes.GO and pathway analysis using the R package GOseq identified many immune-related pathways. Results: RNA sequencing resulted in 11 to 40 million single-end raw reads of 100 bp per sample. After alignment, an average of 80 % of the reads, with 5 % representing multiple mapping, were mapped to the chicken reference transcriptome. Among these detected unique transcripts, there were 2,404 novel genes mainly distributed on chromosomes 1, 2, 4, 3, Z, 5 in decreasing number. Genes were detected as differentially expressed (DE) between treatments (susceptible, resistant, and mock-challenged) at a given time point, or DE between 1 and 5 dpi within the same treatment group. Compared to mock-challenged birds on 5 dpi, susceptible birds exhibited extensive enhancement of their T cell and B cell development, as well as innate and adaptive immune response. However, for the 1 dpi group, susceptible birds exhibited a decreased gene expression of T and B cell development. The differences between the two phenotypes, susceptible and resistant birds, at 5 dpi showed that susceptible birds had increased gene expression of B-cell development and adaptive immune response. Conclusions: This is the first report to our knowledge examining the role of bone marrow cells in responding to APEC infection in broilers. This transcriptome study provides insight and global overview into the response of genes involved in the earliest phases of the immune response to APEC infection. Our data indicate a dynamic interaction between the innate and adaptive immune responses to APEC infection in susceptible birds, providing flexibility and redundancy in the host’s induction of cytokines and chemokines. Additionally, B cell and T cell development are also extensively affected by APEC infection for susceptible birds, resulting in drastic host impairment in early response to infection. The findings of present study shed light on the underlying chicken immune modulation against APEC infection. Also, this study will build a solid foundation for identifying host genetic variation that may be manipulated to enhance resistance to infection and may be useful as colibacillosis control targets.

Project description:Avian pathogenic Escherichia coli (APEC) is considered one of the most common infectious bacterial diseases resulting in significant economic losses in poultry industry worldwide. In order to investigate the association between host immune resistance and miRNA expression in the pathogenic process induced by APEC, miRNA expression profiles in broilers spleen were performed by Solexa deep sequencing from three different treatment groups including non-challenged (NC), challenged-mild pathology (MD), and challenged-severe pathology (SV).In total, 3 462 706, 3 586 689, and 3 591 027 clean reads were obtained for NC, MD, and SV, respectively. After comparing the miRNA expression patterns, 27 differentially expressed miRNAs were identified among the three response groups, which included 13 miRNAs between NC and MD, 17 between NC and SV, and 14 between MD and SV. For these miRNAs, different expression in MD and SV suggested they may have resistance activity in APEC infection. Through integrated analysis of miRNA and mRNA expression patterns, 43 negative pairs between miRNA and mRNA (r < -0.80) were obtained. 4 miRNAs were validated to be significant negatively correlated to targets by quantitative real time PCR: gga-miR-21 (CLEC3B and GGTLA1), gga-miR-429 (TMEFF2, CDC20, SHISA2 and NOX4), gga-miR-146b (LAT2 and WNK1), and gga-miR-215 (C7 and ASL2). Additionally, the expression of gga-miR-21 and gga-miR-146b was significantly up-regulated by LPS induced in HD11 macrophage cell. In contrast, gga-miR-429 has no significant change. In summary, we present the first report that characterized the miRNA profiles of chicken spleen in response to APEC infection, and identified several candidate miRNAs which might accelerate host immune response through down-regulating their specific target genes. Through the intra-air sac route into the left thoracic air sac, 240 non-vaccinated males at 4 weeks of age were challenged with 0.1 ml APEC O1 (10^8 colony forming units) and another 120 non-vaccinated males were non-challenged but treated with 0.1 ml PBS. Detailed information on the APEC O1 strain and challenge process was described by previously described study. Necropsy was performed at 1 day post challenge, and a summarized lesion ranging from 0 to 7 was determined for each APEC-challenged bird. Birds with lesions scoring 0-2 were regarded as mild infection, and those scoring 4-7 were designated as severe infection. The mild and severe pathology meant that birds were resistant and susceptible to APEC infection, respectively. Then, spleens from three groups, consisting of non-challenged, challenged-mild pathology and challenged-severe pathology were subjected to Solexa deep sequencing to investigate the dynamics of chicken miRNA expression.

Project description:BackgroundAvian pathogenic Escherichia coli (APEC) is detrimental to poultry health and its zoonotic potential is a food safety concern. Regulation of antimicrobials in food-production animals has put greater focus on enhancing host resistance to bacterial infections through genetics. To better define effective mechanism of host resistance, global gene expression in the spleen of chickens, harvested at two times post-infection (PI) with APEC, was measured using microarray technology, in a design that will enable investigation of effects of vaccination, challenge, and pathology level.ResultsThere were 1,101 genes significantly differentially expressed between severely infected and non-infected groups on day 1 PI and 1,723 on day 5 PI. Very little difference was seen between mildly infected and non-infected groups on either time point. Between birds exhibiting mild and severe pathology, there were 2 significantly differentially expressed genes on day 1 PI and 799 on day 5 PI. Groups with greater pathology had more genes with increased expression than decreased expression levels. Several predominate immune pathways, Toll-like receptor, Jak-STAT, and cytokine signaling, were represented between challenged and non-challenged groups. Vaccination had, surprisingly, no detectible effect on gene expression, although it significantly protected the birds from observable gross lesions. Functional characterization of significantly expressed genes revealed unique gene ontology classifications during each time point, with many unique to a particular treatment or class contrast.ConclusionsMore severe pathology caused by APEC infection was associated with a high level of gene expression differences and increase in gene expression levels. Many of the significantly differentially expressed genes were unique to a particular treatment, pathology level or time point. The present study not only investigates the transcriptomic regulations of APEC infection, but also the degree of pathology associated with that infection. This study will allow for greater discovery into host mechanisms for disease resistance, providing targets for marker assisted selection and advanced drug development.

Project description:Many reports show an association between the Pst system, the Pho regulon related genes and bacterial virulence. Our previous results showed that a functional Pst system is required for full virulence, resistance to serum, polymyxin B and acid shock However, the interplay between the Pst system and virulence has an unknown molecular basis. To understand global APEC virulent strain responses to Pho regulon activation, we conducted transcriptome profiling experiments comparing the isogenic Pst mutant with its wild type strain chi7122 using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. To understand the global responses to Pho regulon activation of APEC, we use the Affymetrix technology. We conducted transcriptome profiling experiments comparing the APEC chi7122 strain and its isogenic Pst mutant grown in rich phosphate medium using the Affymetrix GeneChipÂ® E. coli Genome 2.0 Array. The Affymetrix GeneChipÂ® E. coli Genome 2.0 Array contains the genome of the E. coli MG1655 and three pathogenic E. coli strain (EDL933, Sakai and CFT073) representing 20,366 genes. While comparing genes expression between Pst mutant and the wild type chi7122 strain, 471 genes are either up- (254) or down-regulated (217) of at least 1.5-fold, with a p-value inferior or equal to 0.05 and a false discovery rate of 2.71%. Experiment Overall Design: RNA extraction was performed onto the chi7122 and K3 strains on four biological replicates. An overnight culture grown at 37Â°C was diluted 100-fold into 5 ml of LB broth and was allowed to grow to mid-log phase (OD600 0,6). Cultures were centrifuged and RNAs were isolated by using the RiboPureâ¢-Bacteria Kit (Ambion, Austin, TX), according to the manufacturerâs recommendations, with the exception that the DNAse 1 treatment was performed twice. RNA extractions were performed on four different days (the chi7122 and K3 RNA were extracted at the same time) and a total of eight hybridizations were performed. Hybridization was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4). GeneChip scan was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4), and data were processed using the FlexArray software (Michal Blazejczyk, Mathieu Miron, Robert Nadon (2007). FlexArray: A statistical data analysis software for gene expression microarrays. Genome Quebec, Montreal, Canada). Raw data were normalized using the RMA algorithm and log2 were generated. The expression value was generated by subtracting the mean value of each replicate of the mutant strain by wild-type strain.

Dataset Information

Deep sequencing-based transcriptome analysis of chicken spleen in response to avian pathogenic Escherichia coli (APEC) infection.

Publications

Deep sequencing-based transcriptome analysis of chicken spleen in response to avian pathogenic Escherichia coli (APEC) infection.

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