P38 MAPK pathway regulates the expression of resistin in porcine alveolar macrophages via Ets2 during Haemophilus parasuis stimulation.

Haemophilus parasuis is a widespread bacterial pathogen causing acute systemic inflammation and leading to the sudden death of piglets. Resistin, a multifunctional peptide hormone previously demonstrated to influence the inflammation in porcine, was extremely increased in H. parasuis-infected tissues. However, the mechanism of resistin expression regulation in porcine, especially during pathogen infection, remains unclear. In the present study, we explored for the first time the transcription factor and signaling pathway mediating the expression of pig resistin during H. parasuis stimulation. We found that H. parasuis induced the expression of pig resistin in a time- and dose-dependent manner via the transcription factor Ets2 in porcine alveolar macrophages during H. parasuis stimulation. Moreover, the expression of Ets2 was mediated by the activation of the p38 MAPK pathway induced by H. parasuis, thus promoting resistin production. These results revealed a novel view of the molecular mechanism of pig resistin production during acute inflammation induced by pathogenic bacteria.

Pathogenicity and Molecular Typing of Fowl Adenovirus-Associated With Hepatitis/Hydropericardium Syndrome in Central China (2015-2018).

In central China, a large number of broiler and layer flocks have suffered from outbreaks of severe hepatitis/hydropericardium syndrome (HHS). This resulted in huge economic losses to the poultry industry, from 2015 to 2018. To identify the specific pathogen and study its pathogenicity, 195 samples from Hubei, Jiangxi, Anhui, Hunan, and Henan provinces in central China were collected. The samples were screened for the adenovirus hexon gene, and neighbor joining was used for the phylogenetic reconstruction of the sequences. Among the collected samples, 122 were found to be positive for fowl adenovirus (FAdV) by PCR, and 73 isolates were obtained. The predominant viral serotype was serotype 4 (FAdV-4), which was found in 48 isolates, while 24 were serotype 10 (FAdV-10), and one was serotype 2 (FAdV-2). The CH/HBTF /1710 isolate was selected for further experiment and inoculated into 33-day-old specific pathogen-free chickens via intramuscular injection or oral administration to evaluate pathogenicity. It was found that the mortality for chickens infected by intramuscular injection or oral administration was 70 and 60%, respectively. Necropsy revealed mild to severe hepatitis and hydropericardium at 5 and 7 days after infection. Ancestor analyses indicated that all of the FAdV-4 strains obtained in this study shared a common Indian precursor and had a close genetic relationship with the JSJ13, SDSX, HN/151025, and SDDM-15 strains common in China.

Haemophilus parasuis Infection Disrupts Adherens Junctions and Initializes EMT Dependent on Canonical Wnt/ß-Catenin Signaling Pathway.

In this study, animal experimentation verified that the canonical Wnt/ß-catenin signaling pathway was activated under a reduced activity of p-ß-catenin (Ser33/37/Thr41) and an increased accumulation of ß-catenin in the lungs and kidneys of pigs infected with a highly virulent strain of H. parasuis. In PK-15 and NPTr cells, it was also confirmed that infection with a high-virulence strain of H. parasuis induced cytoplasmic accumulation and nuclear translocation of ß-catenin. H. parasuis infection caused a sharp degradation of E-cadherin and an increase of the epithelial cell monolayer permeability, as well as a broken interaction between ß-catenin and E-cadherin dependent on Wnt/ß-catenin signaling pathway. Moreover, Wnt/ß-catenin signaling pathway also contributed to the initiation of epithelial-mesenchymal transition (EMT) during high-virulence strain of H. parasuis infection with expression changes of epithelial/mesenchymal markers, increased migratory capabilities as well as the morphologically spindle-like switch in PK-15 and NPTr cells. Therefore, we originally speculated that H. parasuis infection activates the canonical Wnt/ß-catenin signaling pathway leading to a disruption of the epithelial barrier, altering cell structure and increasing cell migration, which results in severe acute systemic infection characterized by fibrinous polyserositis during H. parasuis infection.

Relationship between CYP3A29 and pregnane X receptor in landrace pigs: Pig CYP3A29 has a similar mechanism of regulation to human CYP3A4.

The objective of this study was to provide evidence of the validity of utilizing pigs as a model to study the regulation of human CYP3A4, with special emphasis on drug-drug interactions. We determined the mRNA expression and distribution of CYP3A and metabolic nuclear receptors in different tissues isolated from landrace pigs. Our results showed that CYP3A and metabolic nuclear receptor mRNAs were most highly expressed in liver tissues. The expression of the metabolic nuclear receptor pregnane X receptor (PXR) had a significant correlation with expression of CYP3A29, an analog of human CYP3A4. The correlation between their transcriptional levels was further demonstrated using LPS and TNF-α. The mRNA and protein expression of CYP3A29 and PXR in HepLi cells was significantly reduced by LPS and TNF-α treatment. CYP3A29 promoter activity was dramatically elevated by PXR over expression, whereas LPS and TNF-α treatment inhibited the enhanced CYP3A29 promoter activity that was induced by PXR; presumably through inhibition of PXR promoter activity. Furthermore, the inhibition of CYP3A29 promoter activity by LPS and TNF-α treatment was blocked by knockdown of PXR or retinoid X receptor (RXR). These data suggest high similarity in the regulation mechanism of pig CYP3A29 and human CYP3A4. Our research provided a significant evaluation to determine whether pigs are suitable as an experimental animal model.

Haemophilus parasuis infection activates NOD1/2-RIP2 signaling pathway in PK-15 cells.

Haemophilus parasuis, an important swine pathogen, was recently proven able to invade into endothelial or epithelial cell in vitro. NOD1/2 are specialized NLRs that participate in the recognition of pathogens able to invade intracellularly and therefore, we assessed that the contribution of NOD1/2 to inflammation responses during H. parasuis infection. We observed that H. parasuis infection enhanced NOD2 expression and RIP2 phosphorylation in porcine kidney 15 cells. Our results also showed that knock down of NOD1/2 or RIP2 expression respectively significantly decreased H. parasuis-induced NF-κB activity, while the phosphorylation level of p38, JNK or ERK was not changed. Moreover, real-time PCR result showed that NOD1, NOD2 or RIP2 was involved in the expression of CCL4, CCL5 and IL-8. Inhibition of NOD1 and NOD2 significantly reduced CCL5 promoter activity, even in a more effective way compared with inhibition of TLR.

Neonatal Fc Receptor-Mediated IgG Transport Across Porcine Intestinal Epithelial Cells: Potentially Provide the Mucosal Protection.

It has been well characterized that piglets can absorb colostrum IgG across the intestine to neonatal bloodstream and a certain level of IgG has been found in the mucosal secretions of the porcine intestinal tract. However, little is known about how the maternal IgG transport across the intestinal barrier and how IgG enter the lumen of intestinal tract. In this study, we demonstrated that the porcine neonatal Fc receptor (pFcRn) was expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2) as well as in kidney cells (PK-15), and pFcRn was mainly distributed in the apical side of the polarized IPEC-J2 cells. Analyzing the phylogenetic relatedness of this gene we found that swine and human neonatal Fc receptor (FcRn) amino acid sequence are closer than rodents. We also showed that pFcRn mediated bidirectional IgG transport across polarized IPEC-J2 cells and bound to IgG in a pH-dependent manner. Furthermore, pFcRn-transcytosed viral-specific IgG reduced the transmissible gastroenteritis virus (TGEV) yield from the luminal direction by a 50% tissue culture infective dose (TCID50) assay. Our results indicate that pFcRn-dependent bidirectional IgG transport across the intestinal epithelium plays critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces.

Molecular characterization and functional analysis of duck TRAF6.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role in activating various signaling cascades as an intracellular signal transducer. Although significant progress has been made clarifying TRAF6 function in mammals, the role of TRAF6 in ducks (duTRAF6) remains poorly understood. In the present study, we cloned the full-length duTRAF6 cDNA from duck embryo fibroblasts (DEFs) for the first time. Real-time quantitative reverse transcription-polymerase chain reaction assays showed that duTRAF6 was widely expressed in different tissues. Overexpression of duTRAF6 activated nuclear factor kappa B (NF-κB) and induced interferon-ß expression. Furthermore, a deletion mutant analysis revealed that the duTRAF6 region between aa 115 and 375 was essential for activating NF-κB. In addition, duTRAF6 knockdown by RNA interference significantly reduced poly(I:C)- and Sendai virus-induced NF-κB activation in DEFs. Taken together, our results demonstrate that duTRAF6 plays a crucial immunoregulatory role in the duck innate immune response.

Screening of differentially expressed genes in the growth plate of broiler chickens with tibial dyschondroplasia by microarray analysis.

BACKGROUND: Tibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens. It is characterized by the presence of a non-vascularized and unmineralized cartilage in the growth plate. Previous studies have investigated differential expression of genes related to cartilage development during latter stages of TD. The aim of our study was to identify differentially expressed genes (DEGs) in the growth plate of broiler chickens, which were associated with early stage TD. We induced TD using tetramethylthiuram disulfide (thiram) for 1, 2, and 6 days and determined DEGs with chicken Affymetrix GeneChip assays. The identified DEGs were verified by quantitative polymerase chain reaction (qPCR) assays. RESULTS: We identified 1630 DEGs, with 82, 1385, and 429 exhibiting at least 2.0-fold changes (P < 0.05) at days 1, 2, and 6, respectively. These DEGs participate in a variety of biological processes, including cytokine production, oxidation reduction, and cell surface receptor linked signal transduction on day 1; lipid biosynthesis, regulation of growth, cell cycle, positive and negative gene regulation, transcription and transcription regulation, and anti-apoptosis on day 2; and regulation of cell proliferation, transcription, dephosphorylation, catabolism, proteolysis, and immune responses on day 6. The identified DEGs were associated with the following pathways: neuroactive ligand-receptor interaction on day 1; synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, ether lipid metabolism, JAK-STAT, GnRH signaling pathway, ubiquitin mediated proteolysis, TGF-ß signaling, focal adhesion, and Wnt signaling on day 2; and arachidonic acid metabolism, mitogen-activated protein kinase (MAPK) signaling, JAK-STAT, insulin signaling, and glycolysis on day 6. We validated seven DEGs by qPCR. CONCLUSIONS: Our findings demonstrate previously unrecognized changes in gene transcription associated with early stage TD. The DEGs we identified by microarray analysis will be used in future studies to clarify the molecular pathogenic mechanisms of TD. From these findings, potential pathways involved in early stage TD warrant further investigation.

Effects of high dietary vitamin A supplementation on tibial dyschondroplasia, skin pigmentation and growth performance in avian broilers.

An experiment was conducted to study high dietary vitamin A on tibial dyschondroplasia, growth performance and skin pigmentation in broilers. One hundred and twenty Avian commercial broilers were randomly allotted to three treatments: group C (control group), in which broilers were fed basic diet containing vitamin A 5512IU/kg diet; group A, in which broilers were fed basic diet with addition vitamin A 35512IU/kg; group B, broilers were fed basic diet with supplement vitamin A 65512IU/kg. The experiment lasted 35d and at the end of the trial, broilers were killed and the right proximal tibiotarsi were dissected in longitudinal section for the assessment of TD incidence and TD index, skin from the same area of breast and tibia in broilers were collected to determine pigmentation. The results showed that a high level vitamin A significantly increased the rate of TD incidence and TD index, but middle level vitamin A did not have a significant effect on that. Both low and high retinoic acid decreased growth performance and skin pigmentation in broilers. It suggests that a high dietary vitamin A cause tibial dyschondroplasia in broilers, decreased growth performance and skin pigmentation. It is likely that the effect of vitamin A on TD is mediated through a depression of vitamin D status.