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Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes severe gastroenteritis. The 5'-nucleotidases of pathogens can dephosphorylate adenosine phosphates, boost adenosine levels and suppress the pro-inflammatory immune response. In our previous study, an extracellular nuclease, 5'-nucleotidase, was identified in the extracellular proteins of S. Typhimurium. However, the nuclease activity and the function of the 5'-nucleotidase of S. Typhimurium have not been explored. In the present study, deletion of the 5'-nucleotidase gene is dispensable for S. Typhimurium growth, even under environmental stress. Fluorescence microscopy revealed that the 5'-nucleotidase mutant induced more macrophage extracellular traps (METs) than the wild type did. Furthermore, recombinant 5'-nucleotidase protein (r5Nuc) could degrade λDNA, and the nuclease activity of r5Nuc was optimum at 37 °C and pH 6.0-7.0. The Mg

1. The purpose of this study was to investigate ATP levels and the activities of important enzymes involved in glycolysis and TCA cycle in livers of embryonated chicken eggs infected by infectious bursal disease virus (IBDV).2. Embryonated chicken eggs (9 days) were randomly divided into two groups (50 eggs per group). The first group was inoculated with a very virulent IBDV (vvIBDV) isolate into the chorioallantoic membrane. The second group was maintained as uninfected control eggs and inoculated with physiological saline. Embryo survival was assessed daily, and six embryos were sacrificed at 24, 48, 72, 96, and 120 hpi for examining livers. Viral loads in the livers were evaluated by qRT-PCR. A comparative analysis of markers associated with the regulation of energy metabolism across several functional classes (ATP, pyruvic and lactic acids, mitochondrial protein, NAD+/NADH ratios, and enolase, lactic acid dehydrogenase and the respiratory chain complex I activities) were examined in the context of IBDV infection.3. The results indicated that increases in the enzymatic activities associated with glycolytic metabolism in turn affected the synthesis and cytoplasmic concentrations of ATP at early timepoints in infected chicken embryos. Subsequently, energy metabolism was inhibited through the pathological perturbations of metabolic enzymes and mitochondrial damage, as inferred from reduced ATP generation.4. These results suggested impaired bioenergetics, which may lead to liver dysfunction consequent to IBDV infection, contributing to the disease pathogenesis.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important virulent intracellular pathogen, causes inflammatory gastroenteritis or typhoid. Macrophages play a key role in innate immunity against Salmonella. Salmonella secreted effector K1 (SseK1) encoded by SPI2 has been identified a novel translocated protein. To investigate the role of Salmonella enterica serovar Typhimurium sseK1 about the inflammation and glycolysis in macrophages, the levels of IL-1β, IL-2, IL-4, IL-6, IFN-γ and Nitric Oxide in macrophages infected by S. Typhimurium SL1344 wild-type (WT) group, ΔsseK1 mutant group and sseK1-complemented group were measured. And the glycolysis level was determined in RAW 264.7 cells infected with these different Salmonella strains. The results showed that groups infected by wild-type strain, sseK1 mutant and sseK1-complemented strain upregulated the production of IL-1β, IL-2, IL-4, IL-6, IFN-γ and NO at 3 h, 6 h and 12 h, respectively. The production of IL-1β, IL-2, IL-4, IL-6, IFN-γ and NO in wild-type strain group were significantly decreased compared with the ΔsseK1 mutant group, which suggested that sseK1 down-regulated the production of related inflammatory factors. Moreover, hexokinase, lactic acid and pyruvic acid levels significantly decreased by infection with sseK1 mutant compared to the wild-type strain. The ATP level of ΔsseK1 mutant group was remarkably increased than WT group and sseK1-complemented group. These indicated that the sseK1 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. In summary, the results demonstrated that sseK1 can down-regulate the inflammation-related cytokines and enhance the glycolysis level in macrophages infected by S. Typhimurium, which may be beneficial for S. typhimurium survival in macrophages.

Background Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen that can survive and replicate in macrophages. Glycolysis is essential for immune responses against S. Typhimurium infection in macrophages, and is also associated with apoptosis. S. Typhimurium secreted effector K3 (SseK3) was recently identified as a novel translated and secreted protein. However, there is no study about the role of sseK3 in the relationship between apoptosis and glycolysis in cells infected with S. Typhimurium. It is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in S. Typhimurium-infected macrophages. Results Macrophages were infected with S. Typhimurium SL1344 wild-type (WT), ΔsseK3 mutant or sseK3-complemented strain, and the effects of sseK3 on apoptosis and glycolysis were determined. The adherence and invasion in the ΔsseK3 mutant group were similar to that in the WT and sseK3-complemented groups, indicating that SseK3 was not essential for the adherence and invasion of S. Typhimurium in macrophages. However, the percentage of apoptosis in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. Caspase-3, caspase-8, and caspase-9 enzyme activity in the ΔsseK3 mutant group were significantly lower than in the WT group and sseK3-complemented groups, indicating that sseK3 could improve the caspase-3, caspase-8, and caspase-9 enzyme activity. We also found that there were no significant differences in pyruvic acid levels between the three groups, but the lactic acid level in the ΔsseK3 mutant group was much lower than that in the WT and sseK3-complemented groups. The ATP levels in the ΔsseK3 mutant group were remarkably higher than those in the WT and sseK3-complemented groups. These indicated that the sseK3 enhanced the level of glycolysis in macrophages infected by S. Typhimurium. Conclusions S. Typhimurium sseK3 is likely involved in promoting macrophage apoptosis and modulating glycolysis in macrophages. Our results could improve our understanding of the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3.

Pathogens have evolved an array of strategies to establish a productive infection. The extracellular proteins secreted by pathogens are one of unique mechanisms to evade the host innate immune response. Many secretory proteins transported by the bacterial secretion systems have been widely investigated in Salmonella. Certain extracellular nucleases are essential for bacterial pathogenesis. However, there is no current data available for the enzymatic properties of the proteins secreted by Salmonella. Therefore, in the present study we have identified and characterized the nuclease activity of the extracellular proteins from Salmonella enterica serovar Typhimurium. It was demonstrated that the extracellular proteins from S. Typhimurium exhibited the deoxyribonucleases activity against λDNA by agarose gel electrophoresis and agar plate diffusion method. The activity was observed at 16 °C, 37 °C and 42 °C, and found to be highest at 42 °C and inhibited at temperatures over 60 °C. The nuclease activity was stable under alkaline conditions (pH 7–10) and the optimum pH was 9.0. The nuclease activity was promoted at high ionic strength of Ba2+, Ca2+, Mg2+, and Ni2+. Nuclease zymography analysis revealed that there were four activity bands in the extracellular proteins; followed by LC-ESI/MS/MS analysis seven proteins were identified. As demonstrated by nuclease zymography, the recombinant 5′-nucleotidase protein expressed in the prokaryotic expression system displayed the DNase activity. To our knowledge, the present findings represent the first direct and unambiguous demonstration of the nuclease activity of the extracellular proteins from S. Typhimurium, and it provides an important fundamental for further investigation of the role of the extracellular proteins in pathogenicity and immune evasion.

Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen. Recently, there are increasing researches about the relationship between apoptosis and glycolysis of cells. Previous studies have identified that S. Typhimurium secreted effector K3 (SseK3) is a novel translated and secreted protein. However, there is no study about the role of sseK3 in the relationship between apoptosis and glycolysis of cells infected with S. Typhimurium. It is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in macrophages. Results: The S. Typhimurium SL1344 wild-type (WT) group, ΔsseK3 mutant group and sseK3-complemented group were used to infect macrophages and the effects of sseK3 on apoptosis and glycolysis of macrophages were investigated. The adherence and invasion of ΔsseK3 mutant group for macrophages were similar to WT group and sseK3-complemented group, indicating that SseK3 did not play an important role in the adherence and invasion of S. Typhimurium for macrophages. However, the apoptosis percentage of the ΔsseK3 mutant group was much lower than WT group and sseK3-complemented group using flow cytometry. The caspase-3, caspase-8 and caspase-9 enzyme activity of the ΔsseK3 mutant group were decreased significantly compared with WT group and sseK3-complemented group, which suggested that sseK3 could improve the activities of caspase-3, caspase-8 and caspase-9 enzyme. We also found that the pyruvic acid level did not significantly change among ΔsseK3 mutant group, WT group and sseK3-complemented group, but the lactic acid level of ΔsseK3 mutant group was much lower than WT group and sseK3-complemented group. The ATP level of ΔsseK3 mutant group was remarkably higher than WT group and sseK3-complemented group. These indicated that the sseK3 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. Conclusions: Our data showed that the sseK3 of S. Typhimurium involved in promoting macrophages apoptosis and influencing glycolysis levels of macrophages. These results may give a better clue about the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3.

Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen. Previous studies have identified that S. Typhimurium secreted effector K3 (SseK3) is a novel translated and secreted protein, but it is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in macrophages. Results: The S. Typhimurium SL1344 wild-type (WT) group, ΔsseK3 mutant group and sseK3-complemented group were used to infect macrophages and the effects of sseK3 on apoptosis and glycolysis of macrophages were investigated. The adherence and invasion of ΔsseK3 mutant group for macrophages were similar to WT group and sseK3-complemented group, indicating that SseK3 did not play an important role in the adherence and invasion of S. Typhimurium for macrophages. However, the apoptosis percentage of the ΔsseK3 mutant group was much lower than WT group and sseK3-complemented group using flow cytometry. The caspase-3, caspase-8 and caspase-9 enzyme activity of the ΔsseK3 mutant group were decreased significantly compared with WT group and sseK3-complemented group, which suggested that sseK3 could improve the activities of caspase-3, caspase-8 and caspase-9 enzyme. We also found that the pyruvic acid level did not significantly change among ΔsseK3 mutant group, WT group and sseK3-complemented group, but the lactic acid level of ΔsseK3 mutant group was much lower than WT group and sseK3-complemented group. The ATP level of ΔsseK3 mutant group was remarkably higher than WT group and sseK3-complemented group. These indicated that the sseK3 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. Conclusions: Our data showed that the sseK3 of S. Typhimurium can promote macrophages apoptosis and influence glycolysis levels of macrophages. These results may give a better clue about the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3. Keywords: S. Typhimurium, sseK3, macrophages apoptosis, glycolysis

The objectives of this study are to analyse the pollution status and spatial correlation of soil heavy metals and identify natural and anthropogenic sources of these heavy metals at different spatial scales. Two hundred and twenty-four soil samples (0-20 cm) were collected and analysed for eight heavy metals (Cd, Hg, As, Cu, Pb, Cr, Zn and Ni) in soils of different land-use types in the Yangtze River Delta of Eastern China. The multivariate methods and factorial Kriging analysis were used to achieve the research objectives. The results indicated that the human and natural effects of different land-use types on the contents of soil heavy metals were different. The Cd, Hg, Cu, Pb and Zn in soils of industrial area were affected by human activities, and the pollution level of these heavy metals in this area was moderate. The Pb in soils of traffic area was affected by human activities, and eight heavy metals in soils of residential area and farmland area were affected by natural factor. The ecological risk status of eight heavy metals in soils of the whole study area was light. The heavy metals in soils showed three spatial scales (nugget effect, short range and long range). At the nugget effect and short range scales, the Cd, Hg, Cu, Pb and Zn in soils were affected by human and natural factors. At three spatial scales, the As, Cr and Ni in soils were affected by soil parent materials.

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Histamine is an endogenous nitrogenous compound with extensive effects on immunologic cells and involved in many physiological functions. The current aim was to determine histamine levels in embryonic liver and its association with the pathogenicity of a very virulent infectious bursal disease virus (vvIBDV) isolate serially passaged in chicken embryos. A vvIBDV isolate and the passaged viruses were inoculated into SPF embryonated chicken eggs (0.2 ml per egg) via the chorioallantoic membrane. Embryonic livers were collected at 24, 48, 72, 96, and 120 h post-inoculation and histamine contents were quantified by fluorescence spectrophotometry analyses. Results showed that the histamine content in embryonic livers infected with the original vvIBDV isolate and the early passaged viruses significantly increased 48 h post-inoculation, as compared with the adapted IBDV isolate (p

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.旋毛虫plancitoxin-1-like (Ts-Pt) 是旋毛虫125种DNaseⅡ家族蛋白中唯一具有典型DNaseⅡ活性区域HKD基序的核酸酶，且普遍认为，组氨酸位点是DNaseⅡ的活性氨基酸位点。为研究Ts-Pt活性位点突变体蛋白的核酸酶活性，利用重叠PCR方法获得Ts-Pt活性位点突变体片段，以pET-28a(+)为载体构建重组表达质粒并在大肠杆菌中诱导表达。重组Ts-Pt突变体蛋白经亲和层析纯化后进行SDS-PAGE分析。利用琼脂糖凝胶电泳法和核酸酶酶谱分析重组Ts-Pt突变体蛋白的核酸酶活性。成功构建含Ts-Pt突变体重组质粒的基因工程菌，SDS-PAGE和亲和层析纯化结果显示，重组Ts-Pt突变体蛋白呈包涵体表达。重组蛋白经复性后并没有表现出核酸酶活性，但核酸酶酶谱分析结果显示，包涵体表达的重组Ts-Pt突变体蛋白表现出降解DNA的能力。同时，N端和C端活性位点H及HCK和DHSK突变并不影响Ts-Pt的核酸酶活性，研究结果为进一步研究庞大的DNaseⅡ家族蛋白在旋毛虫发育和感染方面的作用提供一定的参考。.

Interleukin-18 (IL-18) is an important cytokine involved in innate and acquired immunity. In this study, we cloned the full-length chicken IL-18 (ChIL-18) gene from specific-pathogen-free (SPF) chicken embryo spleen cells and provided evidence that the ChIL-18 gene in a recombinant plasmid was successfully expressed in chicken DT40 cells. ChIL-18 significantly enhanced gamma interferon (IFN-γ) mRNA expression in chicken splenocytes, which increased IFN-γ-induced nitric oxide (NO) synthesis by macrophages. The potential genetic adjuvant activity of the ChIL-18 plasmid was examined in chickens by coinjecting ChIL-18 plasmid and inactivated Newcastle disease virus (NDV) vaccine. ChIL-18 markedly elevated serum hemagglutination inhibition (HI) titers and anti-hemagglutinin-neuraminidase (anti-HN)-specific antibody levels, induced the secretion of both Th1- (IFN-γ) and Th2- (interleukin-4) type cytokines, promoted the proliferation of T and B lymphocytes, and increased the populations of CD3+T cells and their subsets, CD3+CD4+and CD3+CD8+T cells. Furthermore, a virus challenge revealed that ChIL-18 contributed to protection against Newcastle disease virus challenge. Taken together, our data indicate that the coadministration of ChIL-18 plasmid and NDV vaccine induces a strong immune response at both the humoral and cellular levels and that ChIL-18 is a novel immunoadjuvant suitable for NDV vaccination.

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus in the Birnaviridae family. Four pathotypes, attenuated, virulent, antigenic variant, and very virulent, have been identified. In this study, we examined the phylogenetic relationship of 25 field isolates that were collected from a single farm during 1989-2008. A sequence analysis of PCR amplified 714 bp VP2 region showed that all the samples were derived from very virulent infectious bursal disease virus (vvIBDV) and were more closely related to the vvIBDV isolate UK661. From 1999, the isolate XA1999 had amino acids I228 and T394. XA2000, XA2001, XA2002, and XA2003-09 had amino acids E279 and T394. From 2004 to 2008, the isolates had amino acids H320, I349, S375, and R381 while the UK661 virus had T228, D279, Q320, V349, P375, K381, and A394. Such mutations do not change key amino acid residues in the domains which are essential for its virulence. It suggests that a virulent IBDV strain could maintain its virulence for a long period in the same chicken farm and the strain is highly stable under normal environments.

To develop a host-vector balanced lethal system of Salmonella typhimurium adenylate cyclase mutant, and detect its biological characteristics.We constructed SL1344ΔcyaΔasd mutant strain by recombinant suicide plasmid (pREAasd), and screened by two-step method, transformed pYA3493 plasmid containing the asd gene without resistance electric into the lack of SL1344 AcyaΔasd, then the recombinant strains SL1344 ΔcyaΔasd (pYA3493) was constructed successfully.The biochemical characteristics and growth rate of the mutant were different from that of the wild strain SL1344, but almost the same as that of the parent strain SL1344Δcya. The mutant strain could neither ferment maltose, lactose, and sorbitol, nor decompose H2S, galactose and rat lee sugar, but still retained the ability to use glucose. The one-day chicken lethal test showed that SL1344ΔcyaΔasd (pYA3493) mutant was at least 104 times lower than SL1344 strain. The protection rate induced by the SL1344ΔcyaΔasd (pYA3493) mutant was 62. 5%.The SL1344ΔcyaΔasd (pYA3493) mutant was successfully constructed, and had good immune protection, it laid a foundation for developing potential oral vaccines.

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica (Bb) challenge.The complete coding sequence (2040 bp) of the prn gene (PRN) and its fragments,5'-terminal 1173 bp fragment (PN),3'-terminal 867 bp fragment (PC), two copies of region I (654 bp; PR I) in PN, and 2 copies of region II (678 bp; PR II) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by isopropyl-beta-D-thiogalactopyranoside. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PR I and GST-2PR II. All five recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PR I group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN,GST-PC or GST-2PR II serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PR I, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5).In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge. Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN. In addition, GST-2PR II and GST-2PR I provided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis on the basis of these five pertactin recombinants.

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. The objective of this study was to evaluate the potential of attenuated Salmonella enterica serovar Choleraesuis C500 strain with a delta asd mutant as an effective live vaccine vector by the Asd+ balanced-lethal host-vector system. Here, we compared the characteristics of S. enterica serovar Choleraesuis delta asdC500 strain with the parent C500 strain, including phenotype, growth rate, virulence, safety, and expression for heterologous antigen. The mean generation times of delta asdC500 mutant, the vector control delta asdC500 (pYA3493), and the parent avirulent C500 vaccine strain in Luria broth were 30.7, 28.1, and 27.9 min, respectively. The fermentation patterns of theses three strains on different carbohydrates, and the levels of production of H2S, were similar. The O and H antigens of delta asdC500 mutant, delta asdC500 (pYA3493) and delta asdC500 (pYA-F1P2) were 6,7:C:1,5, identical to the parent strain C500. By the method of Reed and Muench, groups of mice were challenged by the intraperitoneal route with different amounts of delta asdC500 (pYA3493) or the parent C500 strain, and the virulence of delta asdC500 (pYA3493) with LD50 of 1.1 x 10(7) CFU was a little lower than C500 with LD50 of 4.4 x 10(6) CFU. All piglets inoculated with delta asdC500 (pYA3493) or C500 survived, and no signs of disease were observed during the entire experimental period. No major differences were found in these two groups. In addition, the recombinant pYA-F1P2 plasmid was very stable in the recombinant delta asdC500 (pYA-F1P2) strain, which expressed secretorily a large amount of the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. In this study, we have shown that the delta asdC500 mutant had a series of biological characteristics similar to the parent vaccine strain C500. Furthermore, the strain could express secretorily a large amount of heterologous antigen. It is likely that this Salmonella expression and delivery system could be easily adapted to develop multivalent recombinant Salmonella vaccines against infectious agents using the Asd+ balanced-lethal host-vector system.