Water Extract of Portulaca Oleracea Inhibits PEDV Infection-Induced Pyrolysis by Caspase-1/GSDMD.

Porcine epidemic diarrhea virus (PEDV) belongs to the coronavirus family and the coronavirus genus, causing contact enteric infection in pigs. It is one of the most serious diseases that threatens the pig industry. However, there is currently no specific drug to prevent and treat the disease, indicating that we need to be vigilant about the spread of the disease and the development of anti-PEDV drugs. The dried aerial parts of the plant Portulaca oleracea in the family Portulacaceous, whose decoction can be used to treat acute enteritis, dysentery, diarrhea, and other diseases. This study explored the potential mechanism of water extract of Portulaca oleracea (WEPO) in PEDV-induced pyroptosis in Vero cells. PEDV decreased the viability of Vero cells in a dose- and time-dependent manner, causing cell damage, upregulating the level of intracellular Nlrp3, and inhibiting the level of Gasdermin D (GSDMD) and the activation of Caspase-1. WEPO can inhibit PEDV-induced pyroptosis, reduce the elevation of inflammatory factors caused by infection, and exhibit a dose-dependent effect. Knockdown of Caspase-1 and GSDMD separately can induce the production of the inflammatory factor IL-1ß to significantly decrease and increase, respectively. These results suggest that WEPO can inhibit cell pyroptosis caused by PEDV and that the Caspase-1 and GSDMD pathways play an important role in this process.

Novel pyridinium cationic pleuromutilin analogues overcoming bacterial multidrug resistance.

A series of pyridinium cation-substituted pleuromutilin analogues were designed, synthesized and evaluated for their antibacterial activities in vitro and in vivo. Most derivatives showed potent antibacterial activities, especially e4 that displayed the highest antibacterial activity against multi-drug resistant bacteria and was subjected to time-kill kinetics, resistance studies, cytotoxicity and molecular docking assays. Molecular docking results, scanning electron microscopy and o-nitrophenyl-ß-galactopyranoside tests showed that e4 not only inhibited bacterial protein synthesis but also disrupted bacterial cell walls. Compound e4 showed an ED50 of 5.68 mg/kg against multi-drug resistant Staphylococcus aureus in infected mice model. In in vivo and in vitro toxicity tests, e4 showed low toxic effects with an LD50 of 879 mg/kg to mice. These results suggest that compound e4 may be considered as a new therapeutic candidate for bacterial infections.

Antibiotic resistance and drug modification: Synthesis, characterization and bioactivity of newly modified potent pleuromutilin derivatives with a substituted piperazine moiety.

Antibiotic-resistant bacteria pose a major global public health concern, owing to the lack of effective antibacterial drugs. Consequently, the discovery and development of innovative antibacterial drug classes with unique mechanisms of action are urgently needed. In this study, we designed, synthesised, and tested a series of novel pleuromutilin derivatives with piperazine linker substituted by amino acids moieties to determine their antibacterial properties. Most synthesized compounds exhibited potent activities against Staphylococcus aureus (S. aureus), methicillin-resistant S. aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis. Compound 6l, the most potent antibacterial agent created in this study, displayed a rapid bactericidal activity against MRSA, Klebsiella pneumoniae and S. aureus cfr N12. Moreover, pharmacokinetics study of compound 6l exhibited good PK performance with a low in vivo clearance (CL = 1965 mL/h/kg) and a suitable half-life (T1/2 = 11.614 ± 5.123 h). Molecular docking experiments revealed the binding model of compound 6l to the unmethylated A2503 of peptidyl transferase centre of 23S rRNA. Interaction pattern of 6l with cfr-mediated ribosomes revealed by molecular dynamics. Moreover in vivo mouse systemic infection experiments with compound 6l revealed its effectiveness against MRSA and S. aureus cfr N12 with the ED50 of 11.08 mg/kg and 14.63 mg/kg body weight, respectively.

Hepatotoxic mechanism of diclofenac sodium on broiler chicken revealed by iTRAQ-based proteomics analysis.

BACKGROUND: At the therapeutic doses, diclofenac sodium (DFS) has few toxic side effects on mammals. On the other hand, DFS exhibits potent toxicity against birds and the mechanisms remain ambiguous. OBJECTIVES: This paper was designed to probe the toxicity of DFS exposure on the hepatic proteome of broiler chickens. METHODS: Twenty 30-day-old broiler chickens were randomized evenly into two groups (n = 10). DFS was administered orally at 10 mg/kg body weight in group A, while the chickens in group B were perfused with saline as a control. Histopathological observations, serum biochemical examinations, and quantitative real-time polymerase chain reaction were performed to assess the liver injury induced by DFS. Proteomics analysis of the liver samples was conducted using isobaric tags for relative and absolute quantification (iTRAQ) technology. RESULTS: Ultimately, 201 differentially expressed proteins (DEPs) were obtained, of which 47 were up regulated, and 154 were down regulated. The Gene Ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted to screen target DEPs associated with DFS hepatotoxicity. The regulatory relationships between DEPs and signaling pathways were embodied via a protein-protein interaction network. The results showed that the DEPs enriched in multiple pathways, which might be related to the hepatotoxicity of DFS, were "protein processing in endoplasmic reticulum," "retinol metabolism," and "glycine, serine, and threonine metabolism." CONCLUSIONS: The hepatotoxicity of DFS on broiler chickens might be achieved by inducing the apoptosis of hepatocytes and affecting the metabolism of retinol and purine. The present study could provide molecular insights into the hepatotoxicity of DFS on broiler chickens.

ILDR1 promotes influenza A virus replication through binding to PLSCR1.

As a natural antiviral regulator, phospholipid scramblase 1 (PLSCR1) has been shown to inhibit influenza virus replication in infected cells through interacting with NP of influenza A virus (IAV). But its antiviral function as well as the underlying regulatory mechanism has not been examined in vivo. In the present work, we show that PLSCR1 expression is decreased in H1N1 SIV-infected mice, and Plscr1-/- mice are more susceptible to H1N1 SIV infection. By performing yeast two-hybrid screening, we identified immunoglobulin-like domain-containing receptor 1 (ILDR1) as a novel PLSCR1-binding partner. ILDR1 is highly expressed in the lungs, and its expression level is increased after virus infection. Interestingly, ILDR1 could not directly interact with virus NP protein, but could combine with PLSCR1 competitively. Our data indicates that there is a previously unidentified PLSCR1-ILDR1-NP regulatory pathway playing a vital role in limiting IAV infection, which provides novel insights into IAV-host interactions.

Comparison of the Nutritional Properties and Transcriptome Profiling Between the Two Different Harvesting Periods of Auricularia polytricha.

Auricularia polytricha (A. polytricha), regarded as an edible and medical mushroom, has attracted toward the research interests because of the high nutrition and bioactivity. The nutritional and medical properties of A. polytricha have been well-studied; however, research about the difference of the nutritional properties and transcriptome profiling between the two different harvesting periods of A. polytricha was limited. In this study, the nutritional properties and transcriptome profiling were compared between the two different harvesting periods of A. polytricha: AP_S1 (the stage for the first harvesting period) and AP_S2 (the stage for the third harvesting period). This study showed that AP_S1 had the more growth advantages than AP_S2 including biomass, auricle area and thickness, protein and calcium contents, and most species of the amino acid contents, which contributed to the higher sensory evaluation and acceptability of AP_S1. Transcriptome profiling showed that a total of 30,298 unigenes were successfully annotated in the two different harvesting periods of A. polytricha. At a threshold of two-fold change, 1,415 and 3,213 unigenes were up- and downregulated, respectively. All the differentially expressed genes (DEGs) analysis showed that the some synthesis and metabolic processes were strengthened in AP_S1, especially the synthesis and metabolism of the amino acids and protein. The enhanced energy metabolism pathways could provide more energy for AP_S1 to synthesize the nutritional substance. Moreover, the expressions of 10 selected DEGs involved in the amino acid and protein synthesis pathways and energy metabolism pathways were higher in AP_S1 compared to AP_S2, consistent with Illumina analysis. To the best of our knowledge, this is the first study that compares the nutritional properties and transcriptome profiling between the two different harvesting periods of A. polytricha and the results can present insights into the growth and genetic characteristics of A. polytricha.

Tea Polyphenols Enhanced the Antioxidant Capacity and Induced Hsps to Relieve Heat Stress Injury.

Keap1-Nrf2-ARE and heat shock proteins (Hsps) are important endogenous protection mechanisms initiated by heat stress to play a double protective role for cell adaptation and survival. H9C2 cells and 80 300-day-old specific pathogen-free chickens were randomly divided into the control and tea polyphenol groups and used to establish a heat stress model in vitro and in vivo. This task was conducted to explore the protection and mechanism of tea polyphenols in relieving thermal injury. A supplement with 10 µg/mL tea polyphenols could effectively relieve the heat damage of H9C2 cells at 42°C. Accordingly, weaker granular degeneration, vacuolar degeneration, and nucleus deep staining were shown. A strong antioxidant capacity was manifested in the upregulation of the total antioxidant capacity (T-AOC) (at 5 h, P < 0.05), Hemeoxygenase-1 mRNA (at 2 h, P < 0.01), superoxide dismutase (SOD) (at 2, 3, and 5 h, P < 0.05), and Nrf2 (at 0 and 5 h, P < 0.01). A high expression of Hsps was reflected in CRYAB at 3 h; Hsp27 at 0, 2, and 3 h (P < 0.01); and Hsp70 at 3 and 5 h (P < 0.01). The supplement with 0.2 g/L tea polyphenols in the drinking water also had a good effect in alleviating the heat stress damage of the myocardial cells of hens at 38°C. Accordingly, light pathological lesions and downregulation of the myocardial injury-related indicators (LDH, CK, CK-MB, and TNF-α) were shown. The mechanism was related to the upregulation of T-AOC (at 0 h, P < 0.05), GSH-PX (at 0.5 d, P < 0.01), SOD (at 0.5 d), and Nrf2 (at 0 d with P < 0.01 and 2 d with P < 0.05) and the induced expression of CRYAB (at 0.5 and 2 d), Hsp27 (at 0, 0.5, and 5 d), and Hsp70 (at 0 and 0.5 d). In conclusion, the tea polyphenols enhanced the antioxidant capacity and induced Hsps to relieve heat stress injury.

Antiviral activity of portulaca oleracea L. extracts against porcine epidemic diarrhea virus by partial suppression on myd88/NF-κb activation in vitro.

Porcine epidemic diarrhea virus (PEDV), especially variants, causes a highly contagious enteric disease which could give rise to huge economic losses in the swine industry worldwide. Portulaca oleracea L. has been reported to regulate intestine disease and involved in viral infections. However, the underlying mechanisms of Portulaca oleracea L. extracts against PEDV have not been fully elucidated. In this study, the antiviral effects and potential mechanisms of Portulaca oleracea L. extracts against PEDV were investigated in vitro. We first examined the inhibitory effects of different Portulaca oleracea L. extracts on the PEDV(JX-16 strain) in vitro and found that the water extract of Portulaca oleracea L.(PO)could significantly inhibit PEDV replication by 92.73% on VH cells and 63.07% on Vero cells. Furthermore, time-course analysis showed PO inhibited PEDV replication during the adsorption period of infectious cycle. Western blot and indirect immunofluorescence assay indicated that PO down-regulated the S protein expression in a dose-dependent manner. In addition, our results demonstrated the ability of PO to inhibit PEDV replication in VH cells by down-regulating the cytokine levels (TNF-α,IL-22 and IFN-α) and inhibiting the NF-κB signaling pathway activated by PEDV. Thus, Portulaca oleracea L extracts have potential utility in the preventive and therapeutic strategies for PEDV infection.

A recombinant vaccine of Riemerella anatipestifer OmpA fused with duck IgY Fc and Schisandra chinensis polysaccharide adjuvant enhance protective immune response.

Riemerella anatipestifer (R. anatipestifer) causes septicemia and infectious serositis in domestic ducks, leading to high mortality and great economic losses worldwide. Vaccination is currently considered the best strategy to prevent R. anatipestifer infection in ducklings. In this study, we fused the duck IgY Fc gene to the outer membrane protein A (ompA) of R. anatipestifer. The eukaryotic expression plasmid carrying the fusion gene was transformed into Pichia pastoris (P. pastoris) to express the recombinant ompA and ompA-Fc proteins. Then, the effects of fused Fc on the vitality and antigen processing efficiency of duck peritoneal macrophages (PMø) were evaluated in vitro, whereas their immunogenicity was evaluated in vivo. Furthermore, Schisandra chinensis polysaccharide (SCP) was used to evaluate its immune-conditioning effects on the activation of PMø. SCP was also used as adjuvant to investigate immunomodulation on immunoresponses induced by the fused ompA-Fc in ducklings. The conventional Freund's incomplete adjuvant served as the control of SCP. Notably, ompA-Fc promoted phagocytosis of PMø and significantly increased serum antibody titers, CD4+ and CD8+ T-lymphocyte counts, lymphocyte transformation rate, and serum levels of IL-2 and IL-4. In addition, ducklings injected with the ompA-Fc vaccine exhibited considerably greater resistance to the R. anatipestifer challenge than those that received vaccines based on standalone ompA. Of note, SCP was demonstrated to boost the secretion of nitric oxide (NO), IL-1ß, IL-6, TNF-α, and IFN-ß by duck macrophages. In addition, the supplementation of SCP adjuvant to the ompA-Fc vaccines led to the further enhancement of immune response and vaccine protection. The dose of 200 µg/mL showed the most pronounced effects. This study provided valuable insights into protective strategies against R. anatipestifer infection.

Proteomics analysis of chicken peripheral blood lymphocyte in Taishan Pinus massoniana pollen polysaccharide regulation.

The natural polysaccharides extracted from the pollen of Pinus massoniana (TPPPS) have been shown to be a promising immune adjuvant against several viral chicken diseases. However, the exact mechanism through which TPPPS enhances the host immune response in chicken remains poorly understood. In the current study, chicken peripheral blood lymphocytes were treated with varying concentrations of TPPPS and pro-inflammatory cytokines such as IFN-γ, iIL-2 and IL-6 were measured to determine the optimal dose of the polysaccharide. A comparative analysis was subsequently performed between the proteome of lymphocytes subjected to the best treatment conditions and that of untreated cells. Protein identification and quantitation revealed a panel of three up-regulated and seven down-regulated candidates in TPPPS-treated chicken peripheral blood lymphocytes. Further annotation and functional analysis suggested that a number of those protein candidates were involved in the regulation of host innate immune response, inflammation and other immune-related pathways. We believe that our results could serve as a stepping stone for further research on the immune-enhancing properties of TPPPS and other polysaccharide-based immune adjuvants.

Taishan Pinus massoniana Pollen Polysaccharides Enhance Immune Responses in Chickens Infected by Avian Leukosis Virus Subgroup B.

Immunosuppressive virus, which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks and seriously destroys the poultry industry. Our previous studies have reported that Taishan Pinus massoniana pollen polysaccharide (TPPPS) possess immunomodulatory effects and improve the immune effects of vaccines. In this study, avian leukosis virus subgroup B (ALV-B) was chosen as immunosuppressive virus to artificially establish immunosuppressive models in chickens, and the immune modulatory ability of TPPPS on the immune response of chickens was evaluated. Four randomly assigned groups (Group I-IV) of these immunosuppressed chickens were administered with TPPPS at doses of 0, 100, 200, and 400 mg/kg (every kilogram chick), respectively. Group V was administered with saline as control. At seven day old, 10 chickens randomly selected from Group I-V were inoculated with the attenuated Newcastle disease (ND) vaccine. The results showed that during the monitoring period, TPPPS significantly enhanced weight of immune organs, peripheral lymphocyte proliferation, the percentage of CD4+ and the ratio of CD4+/CD8+, IL-2 and IFN-γ production, and ALV-B antibody positive rate of chickens in a dose-dependent manner, with 400 mg/kg TPPPS being the most effective. In addition, the antibody titer against Newcastle disease virus (NDV) in Group IV with 400 mg/kg was significantly higher than those in other groups. We observed the stronger immunity in the TPPPS group, which indicates that TPPPS could be used as an immunoenhancer to relieve immunosuppression caused by ALV-B in the poultry industry.

Pharmacokinetics and tissue distribution of pefloxacin mesylate in chickens.

A specific, sensitive and stable high-performance liquid chromatography (HPLC)-based analytical method was established to determine the level of pefloxacin mesylate (PM) in the plasma and various tissues of chickens. Chickens were randomly assigned to 12 equal experiment groups, including 11 treatment groups and one control group. The chickens in the treatment groups received oral administration of PM and were sacrificed at different pre-determined time points, with their blood and various organs harvested, extracted and analyzed by HPLC to quantify the level of the residual antibiotic. Method validation studies indicated that the HPLC measurement showed excellent precision, reproducibility, stability and robustness. The obtained pharmacokinetic parameters suggested that PM reached peak levels in various tissues within 1-2 h after its oral administration, and was mainly concentrated in liver and kidney. The antibiotic was also found to be cleared from chicken crureus, brain, testes, ovaries and pancreas at higher rates compared with other organs. Overall, the rapid accumulation of PM could at least be partially attributed to its relatively slow organ clearance. These results could serve as a useful guidance for the rational use of PM and other quinolone-derived antimicrobials in the treatment of infectious diseases in chickens and other animals.

The Taishan Robinia pseudoacacia polysaccharides enhance immune effects of rabbit haemorrhagic disease virus inactivated vaccines.

Robinia pseudoacacia flower, a common component in traditional Chinese medicine, has long been well-known for its high pharmaceutical value. This study aimed to assess the immunopotentiating effects of Taishan Robinia Pseudoacacia polysaccharides (TRPPS) in rabbits inoculated with a rabbit haemorrhagic disease virus (RHDV) inactivated vaccine. The rabbits were administered with the RHDV vaccine in conjunction with varying concentrations of TRPPS, and their blood samples were collected at different time points to analyze the ratio and number of blood lymphocytes. In addition, sera were prepared and analyzed to determine the overall antibody titer and the level of IL-2, a cytokine commonly used as an indicator of immune activity. The various TRPPS-supplemented vaccines were shown to be more effective in enhancing the immune functions of the inoculated rabbits compared to their polysaccharide-free counterpart, with 200 mg/mL of TRPPS exhibiting the most pronounced benefits that were comparable to those of propolis. In addition, the TRPPS-supplemented RHDV inactivated vaccines could significantly improve the survival rates of the immunized rabbits against RHDV infection. Our studies offered convincing experimental evidence for the development of TRPPS as a new type of plant-derived immunopotentiator.

Comparative proteomic analysis provides insight into 10-hydroxy-2-decenoic acid biosynthesis in honey bee workers.

10-Hydroxy-2-decenoic acid (10-HDA) is the major compound produced from the mandibular glands (MGs) of honey bee workers. However, little information is available on the molecular mechanisms of 10-HDA biosynthesis. In our study, based on investigating the 10-HDA secretion pattern and the morphological characteristics of MGs from honey bee workers of different ages, a comparative proteomic analysis was performed in the MGs of workers with different 10-HDA production. In total, 59 up-regulated protein species representing 45 unique proteins were identified in high 10-HDA-producing workers by 2-DE-MALDI-TOF/TOF MS. These proteins were involved in carbohydrate/energy metabolism, fatty acid metabolism, protein metabolism and folding, antioxidation, cytoskeleton, development and cell signaling. Proteins related to fatty acid metabolism, including fatty acid synthase and ß-oxidation enzymes, are potentially crucial proteins involved in 10-HDA biosynthesis pathway. And RNA interference (RNAi) results demonstrated that knockdown of electron transfer flavoprotein subunit beta (ETF-ß), one of the protein related to fatty acid metabolism, decreased 10-HDA production of worker bees, suggesting that ETF-ß was necessary for 10-HDA biosynthesis. This study reveals the characteristics of MGs of worker bees at different developmental stages and proteins associated with 10-HDA biosynthesis, which provides the first insight into the molecular mechanism of 10-HDA biosynthesis.

Taishan Pinus massoniana pollen polysaccharide inhibits subgroup J avian leucosis virus infection by directly blocking virus infection and improving immunity.

Subgroup J avian leucosis virus (ALV-J) generally causes neoplastic diseases, immunosuppression and subsequently increases susceptibility to secondary infection in birds. The spread of ALV-J mainly depends on congenital infection and horizontal contact. Although ALV-J infection causes enormous losses yearly in the poultry industry worldwide, effective measures to control ALV-J remain lacking. In this study, we demonstrated that Taishan Pinus massoniana pollen polysaccharide (TPPPS), a natural polysaccharide extracted from Taishan Pinus massoniana pollen, can significantly inhibit ALV-J replication in vitro by blocking viral adsorption to host cells. Electron microscopy and blocking ELISA tests revealed that TPPPS possibly blocks viral adsorption to host cells by interacting with the glycoprotein 85 protein of ALV-J. Furthermore, we artificially established a congenitally ALV-J-infected chicken model to examine the anti-viral effects of TPPPS in vivo. TPPPS significantly inhibited viral shedding and viral loads in immune organs and largely eliminated the immunosuppression caused by congenital ALV-J infection. Additionally, pre-administration of TPPPS obviously reduced the size and delayed the occurrence of tumors induced by acute oncogenic ALV-J infection. This study revealed the prominent effects and feasible mechanisms of TPPPS in inhibiting ALV-J infection, thereby providing a novel prospect to control ALV-J spread.

Immune-Enhancing Effects of Taishan Pinus massoniana Pollen Polysaccharides on DNA Vaccine Expressing Bordetella avium ompA.

Bordetella avium is the causative agent of bordetellosis, which remains to be the cause of severe losses in the turkey industry. Given the lack of vaccines that can provide good protection, developing a novel vaccine against B. avium infection is crucial. In this study, we constructed a eukaryotic expression plasmid, which expressed the outer membrane protein A (ompA) of B. avium, to prepare a B. avium recombinant ompA-DNA vaccine. Three concentrations (low, middle, and high) of Taishan Pinus massoniana pollen polysaccharides (TPPPS), a known immunomodulator, were used as adjuvants, and their immune conditioning effects on the developed DNA vaccine were examined. The pure ompA-DNA vaccine, Freund's incomplete adjuvant ompA-DNA vaccine, and the empty plasmid served as the controls. The chickens in each group were separately inoculated with these vaccines three times at 1, 7, and 14 days old. Dynamic changes in antibody production, cytokine secretion, and lymphocyte count were then determined from 7 to 49 days after the first inoculation. Protective rates of the vaccines were also determined after the third inoculation. Results showed that the pure DNA vaccine obviously induced the production of antibodies, the secretion of cytokines, and the increase in CD(4+) and CD(8+) T lymphocyte counts in peripheral blood, as well as provided a protective rate of 50% to the B. avium-challenged chickens. The chickens inoculated with the TPPPS adjuvant ompA-DNA vaccine and Freund's adjuvant ompA-DNA vaccine demonstrated higher levels of immune responses than those inoculated with pure ompA-DNA vaccine, whereas only the ompA-DNA vaccine with 200 mg/mL TPPPS completely protected the chickens against B. avium infection. These findings indicate that the B. avium ompA-DNA vaccine combined with TPPPS is a potentially effective B. avium vaccine.

Co-adjuvant effects of plant polysaccharide and propolis on chickens inoculated with Bordetella avium inactivated vaccine.

Taishan Pinus massoniana pollen polysaccharide (TPPPS), propolis (PP) and aloe polysaccharide (AP), used as adjuvants, have been proven to possess immunity-enhancing functions. However, their collaborative immunomodulatory effects are largely unknown. To determine which combination can induce the best effects, the three adjuvants were separately or conjointly added into Bordetella avium inactivated vaccines to investigate their co-adjuvant effects on vaccinated chickens. We found that, among all six adjuvant-treated vaccine inoculated groups (TPPPS, PP, AP, TPPPS-PP, PP-AP and TPPPS-AP), the chickens inoculated with TPPPS, PP or TPPPS-PP adjuvant vaccines showed significantly higher levels of antibody titre, cytokine, lymphocyte transformation and peripheral blood T-lymphocyte count than those of non-adjuvant vaccine inoculated groups (P < 0.05), indicating the good immune-enhancing effects of TPPPS and PP. The TPPPS-PP group showed the highest levels of antibody titres and interleukin-2 (IL-2) at 14-28 days post the first inoculation (dpi), lymphocyte transformation rates (LTRs) at 14-35 dpi, CD4(+) T-lymphocyte counts at 14-42 dpi, and CD8(+) T-lymphocyte counts at 28 dpi. The results revealed that B. avium inactivated vaccine used conjointly with TPPPS and PP induced the strongest humoral and cellular immune responses. Thus, there was a synergistic effect between TPPPS and PP on enhancing immunity, which suggests that they can be used as a novel adjuvant formulation for the development of poultry vaccines.

Characterization and biological activity of Taishan Pinus massoniana pollen polysaccharide in vitro.

Taishan Pinus massoniana pollen polysaccharide (TPPPS) improves cellular and humoral immune responses of animals and is a novel potential immunomodulator. However, the components of TPPPS have not been recognized. To investigate the composition of TPPPS, crude polysaccharide was obtained from Taishan P. massoniana pollen through water extraction and ethanol precipitation. Three homogeneous polysaccharide fractions (TPPPS1, TPPPS2, and TPPPS3) were purified from TPPPS by DEAE-cellulose column chromatography. The average molecular weights of the three polysaccharides were 56, 25, and 128 kDa, respectively. Results of high-performance liquid chromatography (HPLC) showed that TPPPS comprised mannose, ribose, xylose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The biological activity assays showed that TPPPS2 and TPPPS3 significantly promoted spleen lymphocyte proliferation, and that TPPPS3 showed better effect than TPPPS2. TPPPS3 enhanced the secretion of cytokine IL-2 and TNF, whereas TPPPS2 mainly elevated IL-2 secretion. By contrast, TPPPS1 exhibited other effects, and it induced the highest amount of NO production, thereby indicating that TPPPS1 had the best antioxidant activity. TPPPS3 at 50 µg/mL significantly inhibited the proliferation of subgroup B Avian Leukosis virus (ALV-B) through virus adsorption interference in vitro. Results indicated that TPPPS comprised three main components, among which, TPPPS1 mainly showed antioxidant effects, whereas TPPPS2 and TPPPS3 played key roles in immunomodulation, especially TPPPS3. Further studies on the use of a reasonable proportion of TPPPS1-3 may facilitate the development of an effective immunomodulator.

Immunomodulatory effects of Taishan Pinus massoniana pollen polysaccharide and propolis on immunosuppressed chickens.

Co-infection of reticuloendotheliosis virus (REV) and avian leukosis virus subgroup J (ALV-J), which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks in China. Taishan Pinus massoniana pollen polysaccharide (TPPPS) and propolis (PP) have been proven to possess immune modulatory effects and improve the immune effects of vaccines. This study aimed to investigate the immune modulatory ability of TPPPS and PP on chickens co-infected with immunosuppressive viruses. Prior to the study, chickens were artificially established as REV and ALV-J co-infection models. Four randomly assigned groups of these immunosuppressed chickens were successively administered with TPPPS, PP, mixture of TPPPS and PP (TPPPS-PP), or phosphate-buffered saline (PBS) for three days. At nine days old, the four immunosuppressed groups, as well as one normal group, were inoculated with the attenuated Newcastle disease (ND) vaccine. During the monitoring period, the indices of immune organ weight, lymphocyte transformation rates, CD4(+) and CD8(+) T-lymphocyte counts in peripheral blood, IL-2 and IFN-γ secretions, serum antibody titers of ND vaccine, and viral loads in spleens were determined. The results showed that chickens administered with TPPPS, PP, or TPPPS-PP could significantly enhance the levels of the above immune parameters compared to chickens in the PBS group. We observed the strongest immunity in the TPPPS-PP group, which indicates that the combination of TPPPS and PP versus TPPPS or PP alone, could generate better effects on improving the immune system effectiveness of immunosuppressed chickens.

Protective immunity induced by the vaccination of recombinant Proteus mirabilis OmpA expressed in Pichia pastoris.

Proteus mirabilis (P. mirabilis) is a zoonotic pathogen that has recently presented a rising infection rate in the poultry industry. To develop an effective vaccine to protect chickens against P. mirabilis infection, OmpA, one of the major outer membrane proteins of P. mirabilis, was expressed in Pichia pastoris. The concentration of the expressed recombinant OmpA protein reached 8.0µg/mL after induction for 96h with 1.0% methanol in the culture. In addition, OmpA protein was confirmed by SDS-PAGE and Western blot analysis using the antibody against Escherichia coli-expressed OmpA protein. Taishan Pinus massoniana pollen polysaccharide, a known plant-derived adjuvant, was mixed into the recombinant OmpA protein to prepare the OmpA subunit vaccine. We then subcutaneously inoculated this vaccine into chickens to examine the immunoprotective effects. ELISA analysis indicated that an excellent antibody response against OmpA was elicited in the vaccinated chickens. Moreover, a high protection rate of 80.0% was observed in the vaccinated group, which was subsequently challenged with P. mirabilis. The results suggest that the eukaryotic P. mirabilis OmpA was an ideal candidate protein for developing an effective subunit vaccine against P. mirabilis infection.