Huaijun Zhou

Probiotics Stimulate Production of Natural Antibodies in Chickens

ABSTRACT Commensal bacteria in the intestine play an important role in the development of immune response. These bacteria interact with cells of the gut-associated lymphoid tissues (GALT). Among cells of the GALT, B-1 cells are of note. These cells are involved in the production of natural antibodies. In the present study, we determined whether manipulation of the intestinal microbiota by administration of probiotics, which we had previously shown to enhance specific systemic antibody response, could affect the development of natural antibodies in the intestines and sera of chickens. Our findings demonstrate that when 1-day-old chicks were treated with probiotics, serum and intestinal antibodies reactive to tetanus toxoid (TT) and Clostridium perfringens alpha-toxin in addition to intestinal immunoglobulin A (IgA) reactive to bovine serum albumin (BSA) were increased in unimmunized chickens. Moreover, IgG antibodies reactive to TT were increased in the intestines of probiotic-treated chickens compared to those of untreated controls. In serum, IgG and IgM reactive to TT and alpha-toxin were increased in probiotic-treated, unimmunized chickens compared to levels in untreated controls. However, no significant difference in serum levels of IgM or IgG response to BSA was observed. These results are suggestive of the induction of natural antibodies in probiotic-treated, unimmunized chickens. Elucidating the role of these antibodies in maintenance of the chicken immune system homeostasis and immune response to pathogens requires further investigation.

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens

BackgroundRNA-Seq is the recently developed high-throughput sequencing technology for profiling the entire transcriptome in any organism. It has several major advantages over current hybridization-based approach such as microarrays. However, the cost per sample by RNA-Seq is still prohibitive for most laboratories. With continued improvement in sequence output, it would be cost-effective if multiple samples are multiplexed and sequenced in a single lane with sufficient transcriptome coverage. The objective of this analysis is to evaluate what sequencing depth might be sufficient to interrogate gene expression profiling in the chicken by RNA-Seq.ResultsTwo cDNA libraries from chicken lungs were sequenced initially, and 4.9 million (M) and 1.6 M (60 bp) reads were generated, respectively. With significant improvements in sequencing technology, two technical replicate cDNA libraries were re-sequenced. Totals of 29.6 M and 28.7 M (75 bp) reads were obtained with the two samples. More than 90% of annotated genes were detected in the data sets with 28.7-29.6 M reads, while only 68% of genes were detected in the data set with 1.6 M reads. The correlation coefficients of gene expression between technical replicates within the same sample were 0.9458 and 0.8442. To evaluate the appropriate depth needed for mRNA profiling, a random sampling method was used to generate different number of reads from each sample. There was a significant increase in correlation coefficients from a sequencing depth of 1.6 M to 10 M for all genes except highly abundant genes. No significant improvement was observed from the depth of 10 M to 20 M (75 bp) reads.ConclusionThe analysis from the current study demonstrated that 30 M (75 bp) reads is sufficient to detect all annotated genes in chicken lungs. Ten million (75 bp) reads could detect about 80% of annotated chicken genes, and RNA-Seq at this depth can serve as a replacement of microarray technology. Furthermore, the depth of sequencing had a significant impact on measuring gene expression of low abundant genes. Finally, the combination of experimental and simulation approaches is a powerful approach to address the relationship between the depth of sequencing and transcriptome coverage.

Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach

BackgroundMicroRNAs (miRNAs) play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of great concern to human health. The objective of this study was to identify miRNAs associated with AIV infections in chickens.ResultsTotal RNAs were isolated from lung and trachea of low pathogenic H5N3 infected and non-infected SPF chickens at 4 days post-infection. A total of 278,398 and 340,726 reads were obtained from lung and trachea, respectively. And 377 miRNAs were detected in lungs and 149 in tracheae from a total of 474 distinct chicken miRNAs available at the miRBase, respectively. Seventy-three and thirty-six miRNAs were differentially expressed between infected and non-infected chickens in lungs and tracheae, respectively. There were more miRNAs highly expressed in non-infected tissues than in infected tissues. Interestingly, some of these differentially expressed miRNAs, including miR-146, have been previously reported to be associated with immune-related signal pathways in mammals.ConclusionTo our knowledge, this is the first study on miRNA gene expression in AIV infected chickens using a deep sequencing approach. During AIV infection, many host miRNAs were differentially regulated, supporting the hypothesis that certain miRNAs might be essential in the host-pathogen interactions. Elucidation of the mechanism of these miRNAs on the regulation of host-AIV interaction will lead to the development of new control strategies to prevent or treat AIV infections in poultry.

Characterization of a newly developed chicken 44K Agilent microarray

BackgroundThe development of microarray technology has greatly enhanced our ability to evaluate gene expression. In theory, the expression of all genes in a given organism can be monitored simultaneously. Sequencing of the chicken genome has provided the crucial information for the design of a comprehensive chicken transcriptome microarray. A long oligonucleotide microarray has been manually curated and designed by our group and manufactured using Agilent inkjet technology. This provides a flexible and powerful platform with high sensitivity and specificity for gene expression studies.ResultsA chicken 60-mer oligonucleotide microarray consisting of 42,034 features including the entire Mareks disease virus, two avian influenza virus (H5N2 and H5N3), and 150 chicken microRNAs has been designed and tested. In an important validation study, total RNA isolated from four major chicken tissues: cecal tonsil (C), ileum (I), liver (L), and spleen (S) were used for comparative hybridizations. More than 95% of spots had high signal noise ratio (SNR > 10). There were 2886, 2660, 358, 3208, 3355, and 3710 genes differentially expressed between liver and spleen, spleen and cecal tonsil, cecal tonsil and ileum, liver and cecal tonsil, liver and ileum, spleen and ileum (P < 10-7), respectively. There were a number of tissue-selective genes for cecal tonsil, ileum, liver, and spleen identified (95, 71, 535, and 108, respectively; P < 10-7). Another highlight of these data revealed that the antimicrobial peptides GAL1, GAL2, GAL6 and GAL7 were highly expressed in the spleen compared to other tissues tested.ConclusionA chicken 60-mer oligonucleotide 44K microarray was designed and validated in a comprehensive survey of gene expression in diverse tissues. The results of these tissue expression analyses have demonstrated that this microarray has high specificity and sensitivity, and will be a useful tool for chicken functional genomics. Novel data on the expression of putative tissue specific genes and antimicrobial peptides is highlighted as part of this comprehensive microarray validation study. The information for accessing and ordering this 44K chicken array can be found at http://people.tamu.edu/~hjzhou/TAMUAgilent44KArray/

Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation

Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern preimplantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of preimplantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and preimplantation blastocyst growth and development in sheep.

Gene expression profiling in chicken heterophils with Salmonella enteritidis stimulation using a chicken 44 K Agilent microarray

BackgroundSalmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis and usually results from the consumption of contaminated poultry products. The mechanism of SE resistance in chickens remains largely unknown. Previously, heterophils isolated from broilers with different genetic backgrounds (SE-resistant [line A] and -susceptible [line B]) have been shown to be important in defending against SE infections. To dissect the interplay between heterophils and SE infection, we utilized large-scale gene expression profiling.ResultsThe results showed more differentially expressed genes were found between different lines than between infection (SE-treated) and non-infection (control) samples within line. However, the numbers of expressed immune-related genes between these two comparisons were dramatically different. More genes related to immune function were down-regulated in line B than line A. The analysis of the immune-related genes indicated that SE infection induced a stronger, up-regulated gene expression of line heterophils A than line B, and these genes include several components in the Toll-like receptor (TLR) signaling pathway, and genes involved in T-helper cell activation.ConclusionWe found: (1) A divergent expression pattern of immune-related genes between lines of different genetic backgrounds. The higher expression of immune-related genes might be more beneficial to enhance host immunity in the resistant line; (2) a similar TLR regulatory network might exist in both lines, where a possible MyD88-independent pathway may participate in the regulation of host innate immunity; (3) the genes exclusively differentially expressed in line A or line B with SE infection provided strong candidates for further investigating SE resistance and susceptibility. These findings have laid the foundation for future studies of TLR pathway regulation and cellular modulation of SE infection in chickens.

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

BackgroundAvian 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.ResultsTotal 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 2.6 M and 3.3 M 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 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection.ConclusionsA 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.

Progesterone Inhibits Uterine Gland Development in the Neonatal Mouse Uterus

ABSTRACT Uterine glands and their secretions are required for conceptus (embryo/fetus and associated placenta) survival and development. In most mammals, uterine gland morphogenesis or adenogenesis is a uniquely postnatal event; however, little is known about the mechanisms governing the developmental event. In sheep, progestin treatment of neonatal ewes permanently ablated differentiation of the endometrial glands. Similarly, progesterone (P4) inhibits adenogenesis in neonatal mouse uterus. Thus, P4 can be used as a tool to discover mechanisms regulating endometrial adenogenesis. Female pups were treated with sesame vehicle alone as a control or P4 from Postnatal Day 2 (PD 2) to PD 10, and reproductive tracts were examined on PD 5, 10, or 20. Endometrial glands were fully developed in control mice by PD 20 but not in P4-treated mice. All other uterine cell types appeared normal. Treatment with P4 stimulated proliferation of the stroma but suppressed proliferation of the luminal epithelium. Microarray analysis revealed that expression of genes were reduced (Car2, Fgf7, Fgfr2, Foxa2, Fzd10, Met, Mmp7, Msx1, Msx2, Wnt4, Wnt7a, Wnt16) and increased (Hgf, Ihh, Wnt11) by P4 in the neonatal uterus. These results support the idea that P4 inhibits endometrial adenogenesis in the developing neonatal uterus by altering expression of morphoregulatory genes and consequently disrupting normal patterns of cell proliferation and development.

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen.