Gene discovery and expression profiling in porcine Peyer's patch

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.     

Gene expression profiling in embryonic chicken ovary during asymmetric development

The reproductive system in female birds arises as bilateral asymmetrical anlagen, excluding the birds of prey. Earlier, histological and messenger RNA (mRNA) expression profile studies of several genes related to gonadal sex differentiation in chicken embryos tried to elucidate the query of this asymmetry in a scattered manner. To understand the matter precisely, we have focused on mRNA expression of a cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) in second half of the embryonic days (E10–E18). The established role of leptin in development of the embryo and its expression in the embryonic ovary also drove us to check leptin receptor (LEPR) expression in the ovary. Increased expression of FSHR and CYP19A1 in the left ovary compared with that in the right ovary was identified (P < 0.05), promoting preferential left ovarian development and functionality. Significant high expression (P < 0.05) of the apoptotic genes in the right ovary were also involved here. Leptin probably has no direct influence on ovarian asymmetry as no significant variation in gonadal mRNA expression of LEPR was observed within the same experimental days. We propose that asymmetric expression of this cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) leads to the development of dimorphic gonads during embryogenesis.     

Gene expression profiling of peripheral mononuclear cells in lame dairy cows with foot lesions

Lameness is a major health issue and likely the single most common cause of pain and discomfort in dairy cattle. Appropriate treatment is delayed or neglected due, in part, to lack of reliable detection. Assessment of cows with lameness is currently limited to subjective visual scoring systems based on locomotion and posture abnormalities. These systems are unreliable to detect lameness, and therefore, a large number of cows remain undiagnosed. The objective of this research was to search for potential biomarkers for lameness-associated painful inflammatory foot lesions in dairy cattle using microarray-based gene expression profiling of peripheral blood mononuclear cells (PBMC). BOTL5 microarrays spotted in duplicate with cDNA representing bovine immune response genes were interrogated with cDNA samples in an eight-array, balanced complete block design with dye swap. Samples from eight lame cows with inflammatory foot lesions and from eight sound cows were pair-matched by age, weight, days in lactation, and pregnancy status at time of PBMC collection and directly compared with each other on individual arrays. Statistical analysis of resulting fluorescence intensity data revealed 31 genes that were putatively differentially expressed in lame versus sound cows (P < 0.05). Of these, BLASTn analysis and gene ontology information showed that 28 genes had high similarity or homology to known human and/or rodent genes. Validation of 15 of these genes known to be important in inflammation and pain was carried out using relative quantitative real-time RT-PCR, which confirmed the up-regulation of interleukin (IL)-2 (12.68 ± 1.47-fold increase) and IL-10 (2.39 ± 0.55-fold increase), matrix metalloproteinase-13 (MMP-13) (10.44 ± 1.14-fold increase), and chemokine C–C motif receptor-5 (CCR5) (5.26 ± 1.05-fold increase), in lame relative to sound cows (P ≤ 0.05). Similarly, granulocyte-macrophage colony-stimulating factor receptor alpha chain precursor (GM-CSF-R-alpha) (2.30 ± 0.63-fold increase) and IL-4 (2.06 ± 0.59-fold increase) showed a tendency (P = 0.10) for up-regulation in lame compared to sound cows. PBMC co-expression of IL-2, MMP-13, CCR5 and IL-10, and potentially IL-4 and GM-CSF-R-alpha appears to be a promising, objective sign of lameness-related inflammatory foot lesions in dairy cattle. In conclusion, this study revealed potential biomarkers of the presence of foot lesions that could boost diagnostic accuracy of lameness and, ultimately, help identify animals in need of pain relief.     

京海黄鸡Myf5基因表达谱及表达规律

为了探究Myf5基因的表达谱和表达规律,研究其可能的作用机理,本研究根据GenBank上的原鸡(Gallus gallus)Myf5基因cDNA序列,跨内含子设计1对荧光定量引物,采用实时荧光定量PCR技术检测Myf5基因在不同组织的表达情况,绘制其组织表达谱;并选取4个时间点,检测在高表达组织中的表达情况,以研究其表达规律。结果显示,无论是在公鸡还是母鸡中,Myf5基因均在胸肌和腿肌中的表达量最高。在公鸡的其他组织中表达量很低或几乎不表达,母鸡中,除在卵巢中有少量表达外,其他组织中表达量很低或几乎不表达。无论是在公鸡腿肌和胸肌中,还是在母鸡腿肌和胸肌中,Myf5基因的表达量总体呈下降趋势,在16周龄接近体成熟时表达量最低。本研究揭示了京海黄鸡(Gallus gallus)Myf5基因的表达谱以及随肌肉生长发育的表达规律,为进一步研究Myf5基因的作用机理、表达调控以及与MyoG基因相互作用机理奠定基础。     

Gene expression profiling of bovine skeletal muscle in response to and during recovery from chronic and severe undernutrition

Gene expression profiles of LM from beef cattle that underwent significant postweaning undernutrition were studied using complementary DNA (cDNA) microarrays. After 114 d of undernutrition, the RNA from LM showed 2- to 6-fold less expression of many genes from the classes of muscle structural proteins, muscle metabolic enzymes, and extracellular matrix compared with animals on a rapid growth diet. The expression levels of these genes had mostly returned to pretreatment levels after 84 d of realimentation. The gene expression changes associated with undernutrition and BW loss showed an emphasis on downregulation of gene expression specific to fast-twitch fibers, typical of starving mammals, with a preferential atrophy of glycolytic fast-twitch fibers. We also identified a small group of genes that showed 2- to 5-fold elevated expression in LM after 114 d of undernutrition. Putative roles for these genes in atrophying skeletal muscle are regulation of myogenic differentiation (CSRP3), maintenance of mesenchymal stem cells (CYR61), modulation of membrane function (TM4SF2), prevention of oxidative damage (SESN1), and regulation of muscle protein degradation (SQSTM1). A significant increase in stearoyl-CoA desaturase (SCD) gene expression was observed in atrophying muscle, suggesting either that increased fatty acid synthesis is part of the tissue response to caloric restriction, or that SCD plays another role in energy metabolism in the mixed cellular environment of bovine skeletal muscle.     

Gene expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli and Staphylococcus aureus in vitro

Postpartum Dysgalactia Syndrome (PDS) represents a considerable health problem of postpartum sows, primarily indicated by mastitis and lactation failure. The poorly understood etiology of this multifactorial disease necessitates the use of the porcine mammary epithelial cell (PMEC) model to identify how and to what extent molecular pathogen defense mechanisms prevent bacterial infections at the first cellular barrier of the gland. PMEC were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for 3 h and 24 h, in vitro. We focused on differential gene expression patterns of PMEC after pathogen challenge in comparison with the untreated control by performing microarray analysis. Our results show that a core innate immune response of PMEC is partly shared by E. coli and S. aureus. But E. coli infection induces much faster and stronger inflammatory response than S. aureus infection. An immediate and strong up-regulation of genes encoding cytokines (IL1A and IL8), chemokines (CCL2, CXCL1, CXCL2, CXCL3, and CXCL6) and cell adhesion molecules (VCAM1, ICAM1, and ITGB3) was explicitly obvious post-challenge with E. coli inducing a rapid recruitment and activation of cells of host defense mediated by IL1B and TNF signaling. In contrast, S. aureus infection rather induces the expression of genes encoding monooxygenases (CYP1A1, CYP3A4, and CYP1B1) initiating processes of detoxification and pathogen elimination. The results indicate that the course of PDS depends on the host recognition of different structural and pathogenic profiles first, which critically determines the extent and effectiveness of cellular immune defense after infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0178-z) contains supplementary material, which is available to authorized users.     

Flow cytometric immune profiling of specific-pathogen-free chickens before and after infectious challenges

Broilers and layer chickens have been intensively selected for production parameters. This selection has affected immune capacity. Consequently, the fine-tuning of immune responses is becoming important for maximum productivity. Flow cytometry is a recurrent technology used for the immunophenotyping of birds. Studies, however, have focused on the mechanism of specific diseases or have used animals whose immunological condition could be biased-by vaccination or environmental stressors, for example. The aim of this study was to evaluate the immune status of specific-pathogen-free birds across different age ranges to characterize the natural changes that occur over time. Additionally, specific-pathogen-free chickens were challenged with four infectious agents, allowing identification of the subpopulations of peripheral blood immune cells that are consistently altered under various conditions. Several lymphocyte subsets vary naturally with aging, so the interpretation of results using animals of different age ranges must proceed with care. Parameters such as CD8(+)CD28(-), CD8αα(+), CD4(+)CD8(+), and CD8(+)TCRVβ1(+) have been shown to be valuable in understanding immune changes during disease. The use of these data allows a determination of the consistency of cytometric parameters under various conditions, which should ease the interpretation of immunophenotyping and the future application of cytometric analysis in the poultry industry.     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly infectious, oncogenic alpha-herpesvirus known as Marek's disease virus (MDV). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

利用实时定量RT-PCR测定肉鸡组织中CaBP基因的相对表达

试验旨在利用实时定量RT-PCR技术建立肉鸡组织钙结合蛋白(CaBP)基因相对表达量的测定方法,为进一步研究肉鸡钙吸收和利用的分子生物学机理以及骨骼的发育奠定技术基础。根据GenBank中鸡的CaBP基因序列,设计合成引物,进行SYBR GreenⅠ实时定量RT-PCR。以管家基因β-Actin为内参基因,对组织Total RNA进行均一化处理,利用循环阈值(Ct值)的变化计算CaBP基因的相对表达量。结果表明,肉鸡十二指肠及胫骨组织CaBP基因相对表达量分别在27.78~2-4.60和2-2.46~2-17.20范围。表明利用实时定量RT-PCR技术对肉鸡组织CaBP基因相对表达水平进行检测是可行的。     

Gene expression and morphological changes in the intestinal mucosa associated with increased permeability induced by short‐term fasting in chickens

Short‐term fasting for 4.5 and 9 hr has been demonstrated to increase intestinal permeability (IP) in chickens. This study aimed to investigate the effects of 0, 4.5, 9 and 19.5 hr fasting on intestinal gene expression and villus‐crypt architecture of enterocytes in jejunal and ileal samples. On day 38, Ross‐308 male birds were fasted according to their group and then euthanised. Two separate intestinal sections (each 2 cm long, jejunum and ileum) were collected. One section was utilised for villus height and crypt depth measurements. The second section was snap‐frozen in liquid nitrogen for quantitative polymerase chain reaction (qPCR) analysis of tight junction proteins (TJP) including claudin‐1, claudin‐3, occludin, zonula occludens (ZO‐1, ZO‐2), junctional adhesion molecules (JAM) and E‐cadherin. Additionally genes involved in enterocyte protection including glucagon‐like peptide (GLP‐2), heat‐shock protein (HSP‐70), intestinal alkaline phosphatase (IAP), mammalian target of rapamycin (mTOR), toll‐like receptors (TLR‐4), mucin (MUC‐2), cluster differentiation (CD‐36) and fatty acid‐binding protein (FABP‐6) were also analysed. Normally distributed data were analysed using one‐way analysis of variance ANOVA. Other data were analysed by non‐parametric one‐way ANOVA. Villus height and crypt depth were increased (p < .05) only in the ileum after fasting for 4.5 and 9 hr compared with non‐fasting group. mRNA expression of claudin‐3 was significantly reduced in the ileum of birds fasted for 9 and 19.5 hr, suggesting a role in IP modulation. However, all other TJP genes examined were not statistically different from control. Nevertheless, ileal FABP‐6 of all fasted groups was significantly reduced, which could possibly be due to reduced bile acid production during fasting.