Nitrogen (N) fertilization potentially affects soil N mineralization and leaching, and can enhance NH3 volatilization, thus impacting crop production. A fertilizer experiment with five levels of N addition (0, 79, 147, 215 and 375 kg N ha(-1)) was performed in 2009 and 2010 in a maize field in Huanghuaihai region, China, where > 300 kg N ha(-1) has been routinely applied to soil during maize growth period of 120 days. Responses of net N mineralization, inorganic N flux (0-10cm), NH3 volatilization, and maize yield to N fertilization were measured. During the growth period, net N mineralization and nitrification varied seasonally, with higher rates occurring in August and coinciding with the R1 stage of maize growth. Soil NO3--N contributed to more than 60% of inorganic N flux during maize growth. Cumulative NH3 volatilization increased with N additions, with total NH3 volatilization during maize growth accounting for about 4% of added N. Relative to the control, mean maize yield in the fertilizer treatments increased by 17% and 20% in 2009 and 2010, respectively. However, grain yield, aboveground biomass, and plant N accumulation did not increase with added N at levels > 215 kg N ha(-1). These results suggest that the current N rate of 300 kg N ha(-1) is not only excessive, but also reduces fertilizer efficacy and may contribute to environmental problems such as global warming and eutrophication of ground water and streams.

Improved management of soil carbon (C) and nitrogen (N) storage in agro-ecosystems represents an important strategy for ensuring food security and sustainable agricultural development in China. Accurate estimates of the distribution of soil C and N stores and their relationship to crop yield are crucial to developing appropriate cropland management policies. The current study examined the spatial variation of soil organic C (SOC), total soil N (TSN), and associated variables in the surface layer (0-40 cm) of soils from intensive agricultural systems in 19 counties within Henan Province, China, and compared these patterns with crop yield. Mean soil C and N concentrations were 14.9 g kg(-1) and 1.37 g kg(-1), respectively, whereas soil C and N stores were 4.1 kg m(-2) and 0.4 kg m(-2), respectively. Total crop production of each county was significantly, positively related to SOC, TSN, soil C and N store, and soil C and N stock. Soil C and N were positively correlated with soil bulk density but negatively correlated with soil porosity. These results indicate that variations in soil C could regulate crop yield in intensive agricultural systems, and that spatial patterns of C and N levels in soils may be regulated by both climatic factors and agro-ecosystem management. When developing suitable management programs, the importance of soil C and N stores and their effects on crop yield should be considered.

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens. To understand the kinetics and relationships between the humoral (Ab) and antigen specific T cell immunity as well as pathological changes during infectious bronchitis virus (IBV) infection and immunization, one-week-old SPF chickens were vaccinated with live IBV H52 strain and challenged with IBV M41 15 days post primary infection. Chickens were sacrificed every 3 days to monitor antigen specific serum IgG and IBV nucleoprotein-specific immune responses using a chicken MHC I tetramer developed in our laboratory. The results demonstrated that T cell responses developed more rapidly than the humoral (Ab) immune response after vaccination with H52. However, serum IgG dramatically increased after M41 challenge. Chickens from the control, non-vaccinated group developed severe respiratory symptoms and demonstrated significant pathological changes in lung, kidney and bursa of Fabricius post challenge with M41. However, chickens vaccinated with H52 did not demonstrate clinical signs or histological changes post challenge with M41. These results indicated that the live IBV H52 inoculation effectively protected chickens from morbidity and pathological changes associated with IBV infection. These data facilitates the design of a new generation of IBV vaccine.

Although raised-bed planting systems can enhance productivity of summer maize (Zea mays) relative to other systems employed in China, mechanisms for this remain unclear. Because of their potential to enhance maize yield, we examined effects of raise-bed planting on rhizosphere microbial functional groups and associated enzyme activities in 2006 and 2007 crop seasons. Results confirmed that raised-bed planting significantly and substantially increased maize growth. Microbial functional groups and enzyme activities varied during the maize growing season, with patterns often correlated with plant growth. For raised-bed planting, mean numbers of bacteria, fungi, and actinomycetes across all three sampling times in 2006 were 7.3 x 10(6), 1.6 x 10(4), 1.9 x 10(5) CFOs g(-1) dry soil, respectively, 82, 44, and 43% higher than those in flat planting, respectively. In 2007, relative differences were 108, 40, and 34% higher than those in flat planting, respectively. Raised-bed planting yielded mean saccharase, urease, protease and phosphatase activities across sampling times in 2006 of 2.3 mg glucose g(-1) h(-1), 0.8 mg NH3-N g(-1) h(-1), 10.5 mg glycine kg(-1) h(-1), and 0.4 mg nitrophenol g(-1) h(-1), 6, 18, 34, and 31% higher than those in flat planting, respectively. In 2007, relative differences were 10, 16, 35, and 27% higher than those in flat planting, respectively. Rhizosphere enzyme activities were significantly correlated with maize yields. These results suggest that raised-bed planting (1) may enhance maize productivity in part by increasing availability of essential crop nutrients by stimulating microbial activity, and (2) is potentially important in sustainable increasing China's supply of maize. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

This study briefly reviews the formation and development of the discipline of aquatic products quality and safety,and summarizes its characteristics,scope,research areas and directions.We believe that the discipline of the quality and safety of aquatic products is a new focal point of the existing academic disciplines.It is a comprehensive and integrated academic discipline.In view of the wide range of research fields under the discipline,the discipline of aquatic products quality and safety is divided into broad and narrow senses in this study to link up with the other existing aquatic disciplines.The essential issues for constructing this discipline are raised and related recommendations are put forward.

Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2Kd-restricted CTL epitope, and the latter is an H-2Dd-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV.

Interferon gamma (IFN-gamma) is a pleiotropic cytokine that is recognized as an important modulator of the immune response. To date, there is no report that prokaryocyte-derived recombinant equine IFN-gamma has antiviral activity. In this report, the gene coding equine IFN-gamma (EIFN-gamma) mature protein was cloned into pET-28a (+) and the recombinant EIFN-gamma was expressed in Escherichia coli (E. coli). The antiviral activity of expressed recombinant EIFN-gamma was evaluated by using a recombinant Vesicular Stornatits Virus expressing green fluorescence protein (rVSV-GFP) system in the equine fetal kidney-78 cell line (EFK-78). The GFP expression in the EFK-78 cells dramatically decreased in the cells treated with EIFN-gamma in a dose-dependent manner, comparing with the mock-treated cells. The titer of antiviral activity was 1 x 10(3) AU/ml. These results demonstrated that the EIFN-gamma expressed in this study had good biological activity. Pure forms and sufficient quantities of biologically active IFN-gamma could facilitate the study of its activities in modulating immune responses both in vivo and in vitro. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

In this study,the M gene of Porcine reproductive and respiratory syndrome virus(PRRSV) CH-1a strain was amplified from the PRRSV genome by RT-PCR and cloned into the plasmid pSC11.The recombinant plasmid pSC11-PRRSV-M was transfected into TK-143 cells infected with vaccinia virus WR strain.Recombinants vaccinia virus of rWR-PRRSV-M was obtained by selecting blue plaques on the TK-143 cells overlaid with agar containing X-gal,and confirmed by PCR.The M gene expressed in TK-143 cells were identified by SDS-PAGE and confirmed by immunofluorescence assay(IFA).The rWR-PRRSV-M recombinant virus could induce specific humoral immune responses against PRRSV in the immunized mice.

IL-18 is a cytokine originally discovered as an important modulator of immune responses and subsequently shown to be pleiotropic. In this report, we expressed the recombinant equine mature interleukin-18 (rEMIL-18) in E. coli and purified it by nickel affinity gel column chromatography. Purified rEMIL-18 had biological activity commensurate with recombinant human IL-18, as determined by its synergistic effect with recombinant human IL-12 (rhIL-12) on the induction of IFN-γ gene expression in equine peripheral blood mononuclear cells (PBMC). Following intraperitoneal (i.p.) immunization of BALB/c mice with rEMIL-18, nine monoclonal antibodies (mAbs) against equine interleukin-18 (EIL-18) were obtained and characterized. These mAbs recognized different epitopes on equine mature interleukin-18 (EMIL-18) protein based on their reactivity with two peptides containing different amino acid sequences and one of these mAbs has neutralization activity against EIL-18 in an IFN-γ-induction assay.

OBJECTIVE To evaluate the effects of the fusion gene of ubiquitin (Ub) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) M gene on the immune response in inoculated mice. METHODS Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-1a strain, respectively, and the M and Ub gene (U-M) was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression. RESULTS The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M (P<0.05), but lower level of antibody (P<0.05). CONCLUSION Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

Biodiversity is the important material base for human survival and sustainable development.There is biodiversity in forest ecosystem.The biodiversity conservation and forest sustainable development could be realized by the modern forest management measures such as ecosystem management,classification management and natural reserve construction and so on.

The NS1 protein of West Nile virus NY99 strain was amplified by PCR with specific primers and cloned into pMAL-c2X.The recombinant vector was transformed into E.coli TB1 competent cells and a fusion protein MBP-NS1 was expressed after induction with IPTG.The MBP-NS1 protein was purified and analysed by western blot and indirect enzyme-linked immunosorbent assay.The results showed that the purified fusion protein retained good antigenicity and specificity.

In order to investigate the immune responses to the nucleoprotein(NP) of Avian influenza virus,NP gene cut from re-combinant plasmid pVAX-NP was subcloned into a eukaryotic expression vector pCAGGS with an avian-derived promoter.The re-combinant plasmid pCAGGS-NP was obtained after PCR identification and sequencing.Then the positive pCAGGS-NP was subject-ed to transfect eukaryocyte and to immunize SPF chickens.Indirect florescent assay(IFA) was engaged to detect the transient expres-sion of NP,and ELISA was used to detect the serum antibody of the immunized chickens which was resistant to NP protein.The re-sults showed that NP was successfully expressed both in vitro and in vivo and had good immunoreactivity.

The GP5 gene of porcine reproductive and respiratory syndrome virus(PRRSV) CH-1a strain was amplified from the PRRSV genome by RT-PCR,and cloned into the transfer plasmid pSC11 to produce pSC11-GP5.Recombinant vaccinia virus containing the GP5 gene(rWR-PRRSV-GP5) was generated through homologous recombination between pSC11-GP5 and vaccinia virus WR strain in TK-143 cells,purified by plaque assay and identified by PCR.Expression of the GP5 protein was detected by SDS-PAGE.Antigenicity of the recombinant GP5 protein was confirmed by indirect immunofluorescence assay(IFA).Inoculation to mice with rWR-PRRSV-GP5 induced specific humoral immune responses against PRRSV.

For the construction of BF2/peptide tetramer, large amount of recombinant chicken major histocompatibility complex (MHC) Ⅰ heavy chain (BF2-BSP) and light chain Chβ2m were expressed and purified. And a peptide originated from avian Infec- tious bronchitis virus (IBV) nucleoprotein (N71~78) was synthesized. Afterwards, BF2-BSP and Chβ2m were refolded with IBV N71~78 in refolding buffer to generate the monomer of BF2/peptide complex. Then the folded product was treated with enzymatic biotinylation by BirA enzyme and followed by a gel filtration column to purify the BF2/peptide fraction. Finally, the BF2/peptide tetramer was formed by mixing purified, biotinylated monomer with phycoerythrin(PE)-labeled streptavidin at a molar ratio of 5∶1. In order to test if the prepared BF2/peptide tetramer could be employed to detect the efficiency of specific T cell, the peripheral blood mononuclear cells (PBMCs) were isolated from the SPF chickens infected with IBV H52 at 10 days post infection and subjected to flow cytometry stain- ing. The cytotoxitic T lymphocytes (CTLs) response of IBV-infected chicks was evaluated with the prepared BF2/peptide tetramer and 3.65% of CTLs frequency specific to IBV N was detected. All these results indicated that the prepared BF2/peptide tetramer can be used to evaluate the specific T cell immune responses of IBV.

In this study,the VP7 gene of serotype 1 bluetongue virus was amplified by RT-PCR and cloned into prokaryotic expression vector pMAL-c2X.The recombinant plasmid was transformed into E.coli TB1 cells and fusion protein(MBP-VP7) produced after induced with 0.5 mmol/L IPTG.The expressed protein was about 90 ku and existed in soluble form.Indirect ELISA was established using purified MBP-VP7 protein as antigen.

Objective: To label equine IFN-γ monoclonal antibody (McAb) with fluorescein isothiocyanate (FITC) and provide an experimental material for intracellular cytokine staining (ICS) assay of IFN-γ in equine lymphocytes. Methods: Purified equine IFN-γ McAb SB10 was labeled with FITC, then diluted serially and used for ICS assay of IFN-γ in equine PBMCs by flow cytometry. The result was compared with that of flow cytometry by using commercial FITC-labeled bovine IFN-γ McAb CC302. Results: When 106 PBMCs were stained with 1 μl of FITC-CC302, the frequency of PBMCs secreting IFN-γ was 9. 35%. However, when 106 PBMCs were stained with 0. 25 μl of FITC-SB10, the frequency was 9. 90%. Conclusion: The prepared FITC-labeled equine IFN-γ McAb laid a material foundation of developing the methods for immunological study on equine cells and provided a technical platform for monitoring the immune state and investigating the immunological mechanism.

目的:建立检测马白细胞介素-18(equine interleu-kin-18,EIL-18)的双抗体夹心ELISA,为马传染病的免疫学研究提供新方法。方法:将识别不同表位的EIL-18的2株单克隆抗体(mAb)B1G1和S6A3纯化后,利用生物素标记试剂盒对B1G1株mAb进行标记,以重组马白细胞介素-18(rEIL-18)为捕获抗原进行ELISA检测来建立EIL-18双抗体夹心ELISA。结果:经过方阵滴定试验确定捕获抗体的最佳浓度为4mg/L,检测抗体的工作效价为1∶2000。建立的方法可检测马血清中的EIL-18,与猪、牛和羊血清中的IL-18不发生交叉性反应,此方法检测敏感度达到15ng/L,特异性良好。结论:成功建立了EIL-18的双抗体夹心ELISA,为马细胞免疫学研究提供了方法,也为下一步EIL-18ELISA试剂盒的商品化开发奠定了坚实基础。

To develop a multiplex RT-PCR method for simultaneous detection of RVFV,HEV,RV,VSV and FMDV,a set of specific primers were designed based on the published sequences.PCR products of 499 bp,137 bp,274 bp,224 bp and 389 bp were obtained for the respective viruses.The specificity of this method was evaluated by testing representative viruses in the same family and related zoonosis viruses and no PCR products were detected for NDV,AKAV,BEFV and JBEV.This method has a sensitivity of detecting as low as 2.91 ×104 (RVFV),3.14 ×103 (HEV),1.25 ×103 (RV),6.65 ×103 (VSV) and 2.38 ×104 (FMDV) copies of template RNA.Test on 11 RV samples and 7 HEV samples all produced positive results.

To examine the bioactivity of recombinant equine interleukin-18 (rEIL-18) obtained from Escherichia coli, rEIL-18 was evaluated to see if it showed a synergistic effect with recombinant human interleukin-12 (rhIL-12) for the induction of equine interfer-on-γ (EIFN-γ ) gene expression from equine peripheral blood mononuclear cells (PBMCs) by Real-time RT-PCR. REIL-18 could in- duce EIFN-γ mRNA expression from equine PBMCs about 20-fold higher than that of negative control in the presence of rhIL-12. And the effect to induce EIFN-γ gene expression was dose-dependent with rEIL-18 in a fixed concentration of rhIL-12. Nine an-ti-rEIL-18 monoclonal antibodies (mAbs) were also evaluated if they could neutralize the bioactivity of rEIL-18. And one of the 9 mAbs could neutralize rEIL-18 bioactivity and its effect was dose-dependent.