The endogenous development and pathogenicity of Eimeria anseris (Kotlan, 1932) in domestic goslings.

Twenty-one, 25-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 0.5 × 104, 1 × 104, or 100 × 104 sporulated oocysts of Eimeria anseris and sacrificed at intervals from 24 to 216 h post-inoculation (HPI). Nine uninfected goslings served as negative controls. Parts of the visceral organs from goslings, including the intestines, kidneys, and liver, were fixed, sectioned, and observed microscopically. The results revealed that two generations of meronts occurred in the life cycle of E. anseris. The first generation of meronts developed at 24-96 HPI and the second generation at 90-128 HPI. Each meront contained 4-10 merozoites. Development of gamonts began at 128 HPI and mature oocysts appeared at 168 HPI. Developmental stages presented mainly in the epithelial cells of crypts and lamina propria in the posterior parts of the jejunum and ileum. Parasites localized mostly in the cytoplasm and occasionally in the nuclei of host cells. Histological lesions were pronounced in the jejunum and ileum. Desquamation and necrosis of the epithelium of intestine and crypts, infiltration of inflammatory cells, and hemorrhage and mucosal edema were associated with aggregates of endogenous stages. The infected goslings mainly showed severe diarrhea, depression, anorexia, and emaciation, suggesting that E. anseris is highly pathogenic in goslings.

Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

BACKGROUND: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described. METHODS: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration. RESULTS: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains. CONCLUSION: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Efficient inhibition of porcine reproductive and respiratory syndrome virus replication by artificial microRNAs targeting the untranslated regions.

A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5' or 3' UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID(50) (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV.

Salmonella Enteritidis RfbD enhances bacterial colonization and virulence through inhibiting autophagy.

Autophagy is a crucial innate immune response that clears pathogens intracellularly. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. Here, we reported that dTDP-4-dehydro-ß-ւ-rhamnose reductase (RfbD) was able to enhance bacterial colonization in vivo and in vitro by regulating autophagy. We screened the transposon mutant library of Salmonella Enteritidis strain Z11 by High-Content Analysis System, found that rfbD gene has an effect on autophagy. The Z11ΔrfbD-infected group showed greater expression of LC3-II than the Z11-infected group in HeLa, RAW264.7, and J774A.1 cells. Overall, the survival of Z11ΔrfbD in RAW264.7 cells was reduced after 8 h of infection compared to that of the Z11 wild-type strain. In addition, we observed that inhibition of autophagic flux significantly increased the survival of Z11ΔrfbD in RAW264.7 cells. Mice infection experiments revealed that Z11ΔrfbD virulence was significantly reduced, and bacterial load was reduced in the liver and cecum in mice model, and LC3-II expression was significantly increased. These findings indicate an important role of Salmonella Enteritidis protein as a strategy to suppress autophagy and provides new ideas for manipulating autophagy as a novel strategy to treat infectious diseases.

Evaluation of the Protective Immune Response Induced by an rfbG-Deficient Salmonella enterica Serovar Enteritidis Strain as a Live Attenuated DIVA (Differentiation of Infected and Vaccinated Animals) Vaccine in Chickens.

Salmonella enterica serovar Enteritidis (S. Enteritidis), one of the zoonotic pathogens, not only results in significant financial losses for the global poultry industry but also has the potential to spread to humans through poultry and poultry products. Vaccination is an effective method to prevent Salmonella infections. In this study, we constructed a live attenuated DIVA (differentiation of infected and vaccinated animals) vaccine candidate, Z11ΔrfbG, and evaluated its protective effectiveness and DIVA potential in chickens. Compared to that of the virulent wild-type strain, the 50% lethal dose (LD50) of the rfbG mutant strain increased 56-fold, confirming its attenuation. High serum levels of S. Enteritidis-specific IgG titers indicated that a significant humoral immune response was induced in the vaccinated group. After challenge, the nonvaccinated group showed serious clinical symptoms (diarrhea, depression, decreased appetite, ruffled feathers, and weight loss), pathological changes (white nodules in the liver and fatty lesions in liver cells), and death. In contrast, there were no clinical symptoms, pathological changes, or death in the 5 × 106- and 5 × 107-CFU-vaccinated groups. Z11ΔrfbG vaccination significantly reduced S. Enteritidis colonization in the spleen, liver, and cecum. In addition, the Z11ΔrfbG-vaccinated group exhibited a negative response to the serological test, whereas the virulent wild-type Z11 infection group was strongly positive for the serological test, showing a DIVA capability of Z11ΔrfbG vaccination. Overall, our findings demonstrate the viability of the rfbG mutant as a live attenuated chicken vaccine that can discriminate between animals that have been immunized and those that have been infected. IMPORTANCE S. Enteritidis is a highly adapted pathogen that causes significant economic losses in the poultry industry around the world. Vaccination is an effective method of controlling S. Enteritidis infections. Here, we demonstrated that S. Enteritidis Z11ΔrfbG has the potential to be a safe, immunogenic, and DIVA vaccine candidate for the control of Salmonella infections in chickens. Z11ΔrfbG not only provided effective protection in chickens but also distinguished between infected and vaccinated chickens by serological tests.

Characteristic analysis of skin keratinocytes in patients with type 2 diabetes based on the single-cell levels.

BACKGROUND: Keratinocytes play an important role in wound healing; however, less is known about skin keratinocytes in patients with type 2 diabetes mellitus (T2DM). Therefore, this study aimed to search for the transcriptional characteristics of keratinocytes at the single-cell level from T2DM patients, and to provide experimental data for identifying the pathological mechanisms of keratinocytes under pathological conditions. METHODS: We performed single-cell RNA sequencing on the skin tissue from two T2DM patients and one patient without diabetes-induced trauma using the BD Rhapsody™ Single-Cell Analysis System. With the help of bioinformatics R-based single-cell analysis software, we analyzed the results of single-cell sequencing to identify the single-cell subsets and transcriptional characteristics of keratinocytes at the single-cell level, including Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyzes. RESULTS: In this study, we found specific highly expressed signature keratinocyte-related genes. We analyzed the transcriptome of keratinocytes from experimental and control groups and screened a total of 356 differential genes, which were subject to bioinformatics analysis. Enriched pathways included oxidative phosphorylation, antigen processing and presentation, prion and Huntingtons' diseases, bacterial invasion of epithelial cells, thermogenesis, vasopressin-regulated water reabsorption, and protein processing in the endoplasmic reticulum. CONCLUSIONS: This study revealed the characteristics of keratinocytes at the single-cell level and screened a group of differentially expressed genes related to T2DM-associated keratinocytes, oxidative phosphorylation, cytokine receptor interactions, prion diseases, and other signaling pathways.

Occurrence and Characterization of NDM-1-Producing Shewanella spp. and Acinetobacter portensis Co-Harboring tet(X3) in a Chinese Dairy Farm.

Bacteria with carbapenem or tigecycline resistance have been spreading widely among humans, animals and the environment globally, being great threats to public health. However, bacteria co-carrying drug resistance genes of carbapenem and tigecycline in Shewanella and Acinetobacter species remain to be investigated. Here, we detected nine blaNDM-1-carrying Shewanella spp. isolates as well as three A. portensis isolates co-harboring tet(X3) and blaNDM-1 from seventy-two samples collected from a dairy farm in China. To explore their genomic characteristic and transmission mechanism, we utilized various methods, including PCR, antimicrobial susceptibility testing, conjugation experiment, whole-genome sequencing, circular intermediate identification and bioinformatics analysis. Clonal dissemination was found among three A. portensis, of which tet(X3) and blaNDM-1 were located on a novel non-conjugative plasmid pJNE5-X3_NDM-1 (333,311 bp), and the circular intermediate ΔISCR2-tet(X3)-blaNDM-1 was identified. Moreover, there was another copy of tet(X3) on the chromosome of A. portensis. It was verified that blaNDM-1 could be transferred to Escherichia coli C600 from Shewanella spp. by conjugation, and self-transmissible IncA/C2 plasmids mediated the transmission of blaNDM-1 in Shewanella spp. strains. Stringent surveillance was warranted to curb the transmission of such vital resistance genes.

Isolation and Identification of Salmonella in Pork.

Salmonella is considered as one of the most important foodborne zoonotic pathogens that can cause several foodborne diseases and is commonly associated with consumption of meats. Contaminated pork and pork products are major sources of human Salmonella infections in many countries. It is important to investigate and monitor the epidemiology of Salmonella in pork for public health and pork productivity. Here, we describe the method for isolation and identification of Salmonella from pork.

The transcriptional characteristics of mast cells derived from skin tissue in type 2 diabetes patients at the single-cell level.

OBJECTIVES: The mechanisms underlying the role of mast cells in wound healing have not been thoroughly studied, and even fewer data are available on studies related to mast cells in the skin of patients with type 2 diabetes mellitus (T2DM). Therefore, this study aims to explore the transcriptional characteristics of mast cells at the single-cell level in patients with T2DM and provide experimental data for studying mast cell behaviors under abnormal glucose metabolism. METHODS: Two patients with T2DM and one trauma patient without diabetes were enrolled. Samples were derived from skin tissue resected at the time of surgery and were isolated by single cell capture technology on BD platform to prepare single cell cDNA library. Seurat was used to process raw reads and analyze data downstream of single-cell RNA sequencing, including removal of low-quality cells, identification of cell clusters at the single-cell level, and screening for differential genes with fold change > 1.5 and p < 0.05 by two-sided t-test. We performed single-cell RNA sequencing on skin tissues of T2DM patients and non-diabetics and identified the cell cluster of skin, single-cell subsets, and transcriptional characteristics of mast cells at a single-cell level. Meanwhile, gene set enrichment(GSEA) analysis was performed on the differentially expressed genes. RESULTS: A total of 8888 cells were obtained from skin tissue. Clustering analysis revealed eight-cell clusters, identified as smooth muscle cells, dendritic cells, mast cells, and T cells, respectively. Cluster 6 was identified as mast cells with the marker genes TPSAB1, CPA3, TPSB2, MS4A2,KIT, etc., which accounting for 2.7% of the total cell number.Compared with the control group, the genes highly expressed in MCs from T2DM patients, include ADH1C, PAXIP1, HAS1, ARG1, etc., and the low expression genes include PHACTR2, GGA1, RASSF2, etc. GSEA analysis suggested that the signal pathways of MCS in T2DM patients included VEGF signaling pathway, Fc gamma R-mediated phagocytosis, the B cell receptor signaling pathway, natural killer cell-mediated cytotoxicity. CONCLUSIONS: The characteristic genes of MCs in the skin tissues of T2DM patients were described at the single-cell level. These genes and enriched signaling pathways provide a theoretical basis and data support for further researches on dermatopathy in patients with diabetes mellitus.

Psychological Nursing of Patients With Stroke in China: A Systematic Review and Meta-Analysis.

The present study aimed to evaluate the efficacy of psychological nursing of patients with stroke in China. The Embase, PubMed, Cochrane Library, CNKI, and Wanfang databases were searched from inception to February 1, 2020. Randomized controlled trials (RCTs) reporting the efficacy of psychological nursing of patients with stroke were included. Revman 5.3 and Stata 15.0 were used for data analysis. Twelve RCTs and 1,013 patients with stroke were included in this systematic review and meta-analysis. The results revealed a significant difference in the Hamilton depression score between the psychological nursing and usual care groups. The meta-analysis of three studies (n = 235) that used a depressive symptom control of ≥25% as the outcome measure showed a significant difference between the two groups. In addition, significant differences were detected in the National Institute of Health stroke scale score and activities of daily living score between the two groups. The present meta-analysis suggests that in China, compared to the usual care, psychological nursing is more effective for alleviating depressive symptoms, improving neurological rehabilitation, and recovering the ability of daily life.

Analyses of prevalence and molecular typing of Salmonella in the goose production chain.

This study investigated the prevalence of Salmonella and the molecular typing of all isolates in a goose production chain including hatchery, farm, slaughterhouse, and market. A total of 350 Salmonella isolates was detected from 1,030 samples, and 13 serotypes were recovered. The highest Salmonella contamination frequency was observed at the hatchery, which 51.8% (188/363) of samples were Salmonella positive. S. Potsdam and S. Typhimurium were the 2 most common serotypes. S. Potsdam was most frequently found in the hatchery, while S. Typhimurium was widely distributed in the goose production chain. In general, the antibiotic resistance of Salmonella isolates is low, which isolates from the market is comparatively higher than from other production links indicating a possibility of Salmonella cross-contamination in the market. By the multilocus sequence typing (MLST) analysis, 7 different ST types were identified. ST2039 was the most common ST type, which was mostly found from S. Potsdam isolates in hatchery indicating that S. Potsdam might have been long existed in hatchery. The pulsed-field gel electrophoresis (PFGE) analysis of S. Potsdam indicated that S. Potsdam could be transmitted along the production chain. The PFGE analysis of S. Typhimurium showed that PFGE pattern 29 (PF29) was distributed in hatchery, and also in farm and from humans indicating the risk of S. Typhimurium transmitting to humans by the food supply chain. Our study provided the evidence of Salmonella cross-contamination in the slaughterhouse and the retail market of goose production chain, and specific serotypes existed for a long time at a particular production link. The spread of Salmonella along the production chain, might cause harm to humans through cross-contamination. Further studies would be needed to control the Salmonella contamination in hatchery and prevent the transmission of the pathogen during the goose production.

In situ synthesis of biocompatible imidazolium salt hydrogels with antimicrobial activity.

Infection with antibiotic-resistant bacteria is becoming a significant public health risk. In this study, we synthesized a series of imidazolium salt (IMS)-containing polymers and hydrogels and tested their antimicrobial properties against both gram-positive (Staphylococcus aureus and MRSA) and gram-negative (Escherichia coli and PA01) bacteria. IMSs were either grafted as side chains or functionalized in the main chain of linear polymers, which demonstrated antimicrobial properties with minimum inhibitory concentrations as low as 2 µg/mL. Similarly, the optimized IMS-containing hydrogel effectively killed MRSA with a 96.1% killing efficiency and inhibited the growth of PA01. These hydrogels also demonstrated high performance in terms of mechanical property (compressive strength >2 MPa) and were noncytotoxic toward human dermal fibroblasts. STATEMENT OF SIGNIFICANCE: A series of polyimidazolium hydrogels were fabricated with acrylamide monomer and poly(ethylene glycol) dimethacrylate by thermal-initiated polymerization. These hydrogels completely killed methicillin-resistant Staphylococcus aureus and inhibited the growth of Pseudomonas aeruginosa. More importantly, these hydrogels demonstrated adequate mechanical property and biocompatibility. These antimicrobial hydrogels have the potential as biomaterials for preventing infections associated with multidrug-resistant bacteria.

Salmonella Coiled-Coil- and TIR-Containing TcpS Evades the Innate Immune System and Subdues Inflammation.

Toll-like receptors (TLRs) activate innate immunity via interactions between their Toll/interleukin-1 (IL-1) receptor (TIR) domain and downstream adaptor proteins. Here we report that Salmonella Enteritidis produces a secreted protein (TcpS) that contains both a TIR domain and a coiled-coil domain. TcpS blocks MyD88- and TRIF-mediated TLR signaling, inhibits inflammatory responses, and promotes bacterial survival. Early-stage immune evasion by TcpS results in severe tissue damage in the late stage of infection and contributes to Salmonella virulence. TcpS-derived peptides inhibit nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation and reduce lipopolysaccharide (LPS)-elicited systemic inflammation. Therapeutic peptide administration alleviates weight loss of mice infected with H1N1 influenza. Importantly, maximal TcpS-mediated TLR inhibition requires the critical TIR-TcpS residues Y191 and I284, as well as TcpS homodimerization via its N-terminal coiled-coil domain. Our study unveils a mechanism in which TcpS suppresses innate immunity via both its homodimerization and interaction with MyD88. TcpS is also a potential therapeutic agent for inflammation-associated diseases.

Corrigendum: Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1-2 Co-Delivered Intranasally With Flagellin or Polyethyleneimine in Mice and Chickens.

[This corrects the article DOI: 10.3389/fimmu.2017.00326.].

Grafting antibiofilm polymer hydrogel film onto catheter by SARA SI-ATRP.

Catheters are widely used and play an important role in medicine. However, catheter-associated infection is prevalent even under stringent sterile conditions. Biofilms are formed when bacteria populate the surfaces of catheters. This makes the biofilm resistant to antibiotics. Hence, it is imperative for there to be an inherently antifouling and anti-bacterial catheter to mitigate the formation of biofilm. This paper aims to outline the synthesis of non-leachable anti-biofilm and anti-bacterial cationic film coatings through direct polymerization using supplemental activator and reducing agent surface initiated atom transfer radical polymerization (SARA SI-ATRP). Three crosslinked cationic coatings comprising of Diallyl dimethyl ammonium chloride (DADMAC), or ε-poly-L-lysine HCl methacrylic acid (EPL-MA) together with a crosslinker (polyethylene glycol dimethacrylate, PEGDMA) were investigated. These non-leachable covalently linked coatings with DADMAC can achieve more than 2 log reduction (99.0%) with Methicillin-resistant Staphylococcus aureus (MRSA) and 1.25 log reduction (94.4%) with Vancomycin resistant Enterococcus (VRE) in in vitro studies.

Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1-2 Co-Delivered Intranasally with Flagellin or Polyethyleneimine in Mice and Chickens.

Consecutive cases of human infection with H7N9 influenza viruses since 2013 in China have prompted efforts to develop an effective treatment. Subunit vaccines introduced by intranasal administration can block an infection at its primary site; flagellin (fliC) and polyethyleneimine (PEI) have been shown to be potent adjuvants. We previously generated the hemagglutinin (HA)1-2-fliC fusion protein consisting of the globular head domain (HA1-2; amino acids 62-284) of HA fused with Salmonella typhimurium fliC. In the present study, we investigated its effectiveness of both flagellin and PEI as mucosal adjuvants for the H7N9 influenza subunit vaccine. Mice immunized intranasally with HA1-2-fliC and HA1-2-PEI showed higher HA1-2-specific immunoglobulin (Ig)G and IgA titers in serum, nasal wash, and bronchial alveolar lavage fluid. Moreover, splenocyte activation and proliferation and the number of HA1-2-specific interferon (IFN)-γ- and interleukin (IL)-4-producing splenocytes were markedly increased in the fliC and PEI groups; in the latter, there were more cells secreting IL-4 than IFN-γ, suggesting that fliC induced T helper type (Th)1 and Th2 immune responses, and PEI induced Th2-biased responses, consistent with the serum antibody isotype pattern (IgG1/IgG2a ratio). Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving fliC and PEI adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1-2. These findings provide a basis for the development of H7N9 influenza HA1-2 mucosal subunit vaccines.

HA1-2-fljB Vaccine Induces Immune Responses against Pandemic Swine-Origin H1N1 Influenza Virus in Mice.

In 2009, a novel pandemic swine-origin influenza A (H1N1) virus caused a public emergency of international concern. Vaccination is the primary strategy for the control of influenza epidemics. However, the poor immunopotency of many vaccine antigens is a major barrier to the development of effective vaccines against influenza. Flagellin, a Toll-like receptor 5 (TLR5) ligand, has been used as an adjuvant to enhance the immunopotency of vaccines in preclinical studies. Here, we developed a recombinant candidate vaccine, HA1-2-fljB, in which the globular head of the hemagglutinin (HA) antigen (residues 62-284) from H1N1 virus was fused genetically to the N-terminus of Salmonella typhimurium fljB. The recombinant HA1-2-fljB protein was expressed efficiently in Escherichia coli, and the immunogenicity and protective efficacy of recombinant HA1-2-fljB were evaluated in a mouse model. Immunization with HA1-2-fljB elicited robust IgG antibodies and neutralizing antibodies and completely protected the mice against infection by swine-origin influenza A/swine/Jangsu/38/2010 (H1N1). These results suggest that HA antigen placed at the N-terminus of flagellin is also an excellent starting point for creating a fusion HA1-2-fljB protein as a candidate vaccine, and the recombinant HA1-2-fljB protein will contribute to the development of a more effective vaccine against swine-origin influenza virus infection.

Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo.

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region. The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyleneimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis. The results showed that expression of pEGFP-N1 was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the beta-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

A porcine reproductive and respiratory syndrome virus vaccine candidate based on PRRSV glycoprotein 5 and the Toll-like receptor 5 agonist Salmonella typhimurium flagellin.

Glycoprotein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV) is a key inducer of neutralizing antibodies. A truncated GP5 gene lacking the signal peptide and transmembrane sequences was amplified via an overlap PCR method and inserted into prokaryotic expression vectors, pET32a or pGEX-6p-1, to add an His or GST tag, respectively. His-tagged GP5 was induced with IPTG, verified by SDS-PAGE and Western blotting, and purified to serve as an immunogen accompanied with the Salmonella typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. Levels of TLR5 and cytokine mRNAs in spleens of mice following injection with FliC were detected by qRT-PCR to verify the activation of innate immunity. FliC was used as an adjuvant and administered with the GP5 to C57BL/6 mice via intraperitoneal injection. Coadministration of GP5 with FliC induced a significantly enhanced GP5-specific IgG and IFN-γ response compared with administration of GP5 alone, and the GP5-specific titer in the GP5 + FliC coadministration group was elevated almost twofold after the third immunization. These results indicate that FliC is an effective adjuvant, increasing the induction of antibodies against GP5 with the induction of both humoral and cellular immune responses.

[Construction and screening of the artificial miRNA plasmids targeting porcine Toll-like receptor 7 gene].

OBJECTIVE: To construct and screen specific artificial microRNA (amiRNA) expression plasmids targeting porcine Toll-like receptor 7 (TLR7) gene. METHODS: The 1984-2649 bp sequence of the porcine TLR7 cDNA was amplified by RT-PCR and inserted into plasmid pEGFP-N1 to construct the fusion expression vector pTLR7-EGFP. Five amiRNAs targeting porcine TLR7 gene were designed and cloned into pcDNA5-miR to construct the recombinant interfering plasmids pcDNA5-miRTLR7. NIH-3T3 cells were co-transfected with plasmids pTLR7-EGFP and pcDNA5-miRTLR7. The expression levels of amiTLR7 were monitored by RT-PCR and their silencing efficiencies were detected by fluorescent microscopy and flow cytometry. RESULTS: All five amiTLR7s were successfully constructed and could effectively silence the expression of TLR7 gene in NIH-3T3 cells with the inhibition efficiencies ranging from 36.99% to 97.28%, among which amiTLR7-3 had the best interference efficiency. CONCLUSION: The specific artificial amiRNA expression plasmids targeting porcine TLR7 gene have been successfully constructed, and the optimal amiTLR7 with the highest inhibition efficiency has been screened.