Development of multiplex PCR for simultaneous detection and differentiation of six DNA and RNA viruses from clinical samples of sheep and goats.

Multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and subsequently evaluated for its effectiveness in detecting simultaneously single and mixed infections in sheep and goats. Specific primers for three DNA viruses and three RNA viruses, including foot and mouth disease virus (FMDV), Bluetongue virus (BTV), peste des petits ruminants virus (PPRV), sheeppox virus (SPPV), goatpox virus (GTPV) and orf virus (ORFV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive because it could detect at least 100pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty seven clinical samples collected from sheep and goats were detected among forty three samples tested by both uniplex and multiplex PCR, showing highly identification. As results of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in sheep and goats with a reaction.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effects of a recombinant vaccine against Aeromonas hydrophila.

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for recombinant Aeromonas hydrophila vaccine administration via bath or injection in juvenile grass carp were studied. The results showed that SWCNT as a vector for the recombinant protein aerA, augmented the production of specific antibodies, apparently stimulated the induction of immune-related genes, and induced higher level of survival rate compared with free aerA subunit vaccine. Furthermore, we compared the routes of bath and intramuscular injection immunization by SWCNTs-aerA vaccine, and found that similar antibody levels induced by SWCNTs-aerA were observed in both immunization routes. Meanwhile, a similar relative percentage survival (approximately 80%) was found in both a 40 mg/L bath immunization group, and a 20 µg injection group. The results indicate that functionalized SWCNTs could be a promising delivery vehicle to potentiate the immune response of recombinant vaccines, and might be used to vaccinate juvenile fish by bath administration method.

[The new method monitoring crop water content based on NIR-Red spectrum feature space].

Moisture content is an important index of crop water stress condition, timely and effective monitoring of crop water content is of great significance for evaluating crop water deficit balance and guiding agriculture irrigation. The present paper was trying to build a new crop water index for winter wheat vegetation water content based on NIR-Red spectral space. Firstly, canopy spectrums of winter wheat with narrow-band were resampled according to relative spectral response function of HJ-CCD and ZY-3. Then, a new index (PWI) was set up to estimate vegetation water content of winter wheat by improveing PDI (perpendicular drought index) and PVI (perpendicular vegetation index) based on NIR-Red spectral feature space. The results showed that the relationship between PWI and VWC (vegetation water content) was stable based on simulation of wide-band multispectral data HJ-CCD and ZY-3 with R2 being 0.684 and 0.683, respectively. And then VWC was estimated by using PWI with the R2 and RMSE being 0.764 and 0.764, 3.837% and 3.840%, respectively. The results indicated that PWI has certain feasibility to estimate crop water content. At the same time, it provides a new method for monitoring crop water content using remote sensing data HJ-CCD and ZY-3.

[Plasma concentration and pharmacokinetics of ursolic acid carried in self-microemulsifying drug delivery system in rats studied by UPLC-MS/MS].

This study is to report the establishment of an UPLC-MS/MS method for the determination of plasma concentration of UA carried in self-microemulsifying drug delivery system (SMEDDS) and its pharmacokinetics in rats. It was used for determination and analysis when serum with internal standard was extracted from C18 solid-phase column. Acquity UPLC BEH C18 column (100 mm x 2.1 mm, 1.7 microm) was used for separation. The mobile phase was acetonitrile -0.1% ammonia with gradient elution at the flow rate of 0.2 mL x min(-1). The column temperature was 40 degrees C and the detection wave length was 210 nm. It was detected by negative ion using electrospray ionization source (ESI) and scanned by multiple reaction ion monitoring (MRM) mode. The liner relationship of UA was very good in the range of 1.19-3 815.00 ng x mL(-1) (r = 0.999 0). Recovery rate of different concentrations were 87.42%-89.95%. The precision of inter-day and intra-day were less than 11%. The method developed in our study was proved to be sensitive, rapid and simple. It is suitable for the pharmacokinetic study of UA-SMEDDS in rats.

Assessment of the AquaCrop model for use in simulation of irrigated winter wheat canopy cover, biomass, and grain yield in the North China Plain.

Improving winter wheat water use efficiency in the North China Plain (NCP), China is essential in light of current irrigation water shortages. In this study, the AquaCrop model was used to calibrate, and validate winter wheat crop performance under various planting dates and irrigation application rates. All experiments were conducted at the Xiaotangshan experimental site in Beijing, China, during seasons of 2008/2009, 2009/2010, 2010/2011 and 2011/2012. This model was first calibrated using data from 2008/2009 and 2009/2010, and subsequently validated using data from 2010/2011 and 2011/2012. The results showed that the simulated canopy cover (CC), biomass yield (BY) and grain yield (GY) were consistent with the measured CC, BY and GY, with corresponding coefficients of determination (R(2)) of 0.93, 0.91 and 0.93, respectively. In addition, relationships between BY, GY and transpiration (T), (R(2) = 0.57 and 0.71, respectively) was observed. These results suggest that frequent irrigation with a small amount of water significantly improved BY and GY. Collectively, these results indicate that the AquaCrop model can be used in the evaluation of various winter wheat irrigation strategies. The AquaCrop model predicted winter wheat CC, BY and GY with acceptable accuracy. Therefore, we concluded that AquaCrop is a useful decision-making tool for use in efforts to optimize wheat winter planting dates, and irrigation strategies.

[Estimating canopy water content in wheat based on new vegetation water index].

Moisture content is an important indicator for crop water stress condition, timely and effective monitoring crop water content is of great significance for evaluate crop water deficit balance and guide agriculture irrigation. In order to improve the saturated problems of different forms of typical NDWI (Normalized Different Water Index), we tried to introduce EVI (Enhanced Vegetation Index) to build new vegetation water indices (NDWI#) to estimate crop water content. Firstly, PROSAIL model was used to study the saturation sensitivity of NDWI, and NDWI# to canopy water content and LAI (Leaf Area Index). Then, the estimated model and verified model were estimated using the spectral data and moisture data in the field. The result showed that the new indices have significant relationships with canopy water content. In particular, by implementing modified standardized for NDWI1450, NDWI1940, NDWI2500. The result indicated that newly developed indices with visible-infrared and shortwave infrared spectral feature may have greater advantage for estimation winter canopy water content.

[New vegetation index fusing visible-infrared and shortwave infrared spectral feature for winter wheat LAI retrieval].

Considering the great relationships between shortwave infrared (SWIR) and leaf area index (LAI), innovative indices based on water vegetation indices and visible-infrared vegetation indices were presented. In the present work, PROSAIL model was used to study the saturation sensitivity of new vegetation indices to LAI. The estimate models about LAI of winter wheat were built on the basis of the experiment data in 2009 acting as train sample and their precisions were evaluated and tested on the basis of the experiment data in 2008. Ten visible-infrared vegetation indices and five water vegetation indices were used to construct new indices. The result showed that newly developed indices have significant relationships with LAI by numerical simulations and in-situ measurements. In particular, by implementing modified standardized LAI Determining Index (sLAIDI *), all new indices were neither sensitive to water variations nor affected by saturation at high LAI levels. The evaluation models could improve prediction accuracy and have well reliability for LAI retrieval. The result indicated that visible-infrared vegetation indices combined with water index have greater advantage for LAI estimation.

[Estimation of winter wheat protein content based on new indexes].

Wheat protein content is an important indicator often employed in wheat sale price. Spectral indexes and concurrent winter wheat protein content (WWPC) samples were obtained across three years. Data from 2008/2009 and 2009/2010 were utilized to build the new ratio indexes and product indexes, and then selected grey relational method and partial least squares method were used to improve the estimation accuracy of WWPC, data from 2011/2012 was utilized to validate model. The results showed that the correlation coefficients between ratio indexes and WWPC were better than that between single indexesand WWPC. The r of single indexes and ratio indexes were 0.726 and 0.751, respectively, and the product indexes were used to improve the parts of single indexes. The estimation accuracy of WWPC was improved by using GRA-PLS, the determination coefficients (R2) of single indexes, ratio indexes and product indexes were 0.537, 0.631 and 0.521, respectively, and corresponding root mean square errors (RMSE) were 0.665%, 0.564% and 0.574%, respectively. The results indicated that it was feasible to estimate WWPC by building new ratio indexes and product indexes, and then applying the GRA-PLS.

[Band depth analysis and partial least square regression based winter wheat biomass estimation using hyperspectral measurements].

The major limitation of using existing vegetation indices for crop biomass estimation is that it approaches a saturation level asymptotically for a certain range of biomass. In order to resolve this problem, band depth analysis and partial least square regression (PLSR) were combined to establish winter wheat biomass estimation model in the present study. The models based on the combination of band depth analysis and PLSR were compared with the models based on common vegetation indexes from the point of view of estimation accuracy, subsequently. Band depth analysis was conducted in the visible spectral domain (550-750 nm). Band depth, band depth ratio (BDR), normalized band depth index, and band depth normalized to area were utilized to represent band depth information. Among the calibrated estimation models, the models based on the combination of band depth analysis and PLSR reached higher accuracy than those based on the vegetation indices. Among them, the combination of BDR and PLSR got the highest accuracy (R2 = 0.792, RMSE = 0.164 kg x m(-2)). The results indicated that the combination of band depth analysis and PLSR could well overcome the saturation problem and improve the biomass estimation accuracy when winter wheat biomass is large.

Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses.

Uniplex and multiplex reverse transcription-polymerase chain reaction (RT-PCR) and PCR protocols were developed and evaluated subsequently for its effectiveness in detecting simultaneously single and mixed infections in swine. Specific primers for three DNA viruses and three RNA viruses, including classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) were used for testing procedure. A single nucleic acid extraction protocol was adopted for the simultaneous extraction of both RNA and DNA viruses. The multiplex PCR consisted with two-step procedure which included reverse transcription of RNA virus and multiplex PCR of viral cDNA and DNA. The multiplex PCR assay was shown to be sensitive detecting at least 450pg of viral genomic DNA or RNA from a mixture of six viruses in a reaction. The assay was also highly specific in detecting one or more of the same viruses in various combinations in specimens. Thirty clinical samples and aborted fetuses collected from 4- to 12-week-old piglets were detected among 39 samples tested by both uniplex and multiplex PCR, showing highly identification. Because of the sensitivity and specificity, the multiplex PCR is a useful approach for clinical diagnosis of mixed infections of DNA and RNA viruses in swine.

Oral vaccination with attenuated Salmonella enterica serovar Typhimurium expressing Cap protein of PCV2 and its immunogenicity in mouse and swine models.

Attenuated Salmonella enterica serovar Typhimurium (S. typhimurium) was selected as a transgenic vehicle for the development of oral vaccines against Porcine circovirus type 2 (PCV2). The Cap-encoding gene of PCV2 was amplified by PCR and cloned into expression vector pYA3341. The recombinant plasmid pYA3341-Cap was transformed into attenuated S. typhimurium X4550. BALB/c mice were inoculated orally with various doses of attenuated S. typhimurium X4550/pYA3341-Cap. The bacterium was safe to mice at dose of 2×10(9)cfu and eventually eliminated in the spleen and mesenteric lymph nodes at 4 weeks post-immunization. The flow cytometry analysis showed that the percentage of CD4(+) T cells and CD4(+)/CD8(+) ratio were increased significantly in mice immunized with attenuated S. typhimurium X4550/pYA3341-Cap. Vaccine tests in swine showed that the oral immunization with attenuated S. typhimurium X4550/pYA3341-Cap could elicit significantly higher Cap antibody titers in the treated swine than the control groups. Virus neutralization test showed that serum from the swine treated with attenuated S. typhimurium X4550/pYA3341-Cap had significant levels of neutralization activities. The swine lymphocyte proliferative responses indicated that attenuated S. typhimurium X4550/pYA3341-Cap could induce obvious cellular immune response. An in vivo challenge study showed the swine treated with attenuated S. typhimurium X4550/pYA3341-Cap had significantly lower PCV2-associated lesions and PCV2 viremia than the control groups. The results indicated that attenuated S. typhimurium X4550/pYA3341-Cap can be a potential vaccine against PCV2 infections.

Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections.

[Hyperspectral estimation of leaf water content for winter wheat based on grey relational analysis (GRA)].

The objective of the present study was to compare two methods for the precision of estimating leaf water content (LWC) in winter wheat by combining stepwise regression method and partial least squares (SRM-PLS) or PLS based on the relational degree of grey relational analysis (GRA) between water vegetation indexes (WVIs) and LWC. Firstly, data utilized to analyze the grey relationships between LWC and the selected typical WVIs were used to determine the sensitivity of different WVIs to LWC. Secondly, the two methods of estimating LWC in winter wheat were compared, one was to directly use PLS and the other was to combine SRM and PLS, and then the method with the highest determination coefficient (R2) and lowest root mean square error (RMSE) was selected to estimate LWC in winter wheat. The results showed that the relationships between the first five WVI and LWC were stable by using GRA, and then LWC was estimated by using PLS and SRM-PLS at the whole stages with the R2 and RMSEs being 0.605 and 0.575, 4.75% and 7.35%, respectively. The results indicated that the estimation accuracy of LWC could be improved by using GRA firstly and then by using PLS and SRM-PLS.

Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.

[A field-based pushbroom imaging spectrometer for estimating chlorophyll content of maize].

As an image-spectrum merging technology, the field-hperspectral imaging technology is a need for dynamic monitoring and real-time management of crop growth information acquiring at field scale in modern digital agriculture, and it is also an effective approach to promoting the development of quantitative remote sensing on agriculture. In the present study, the hyperspectral images of maize in potted trial and in field were acquired by a self-development push broom imaging spectrometer (PIS). The reflectance spectra of maize leaves in different layers were accurately extracted and then used to calculate the spectral vegetation indices, such as TCARI, OSAVI, CARI and NDVI. The spectral vegetation indices were used to construct the prediction model for measuring chlorophyll content. The results showed that the prediction model constructed by spectral index of MCARI/OSAVI had high accuracy. The coefficient of determination for the validation samples was R2 = 0.887, and RMSE was 1.8. The study indicated that PIS had extensive application potentiality on detecting spectral information of crop components in the micro-scale.

[Study on relationship between new characteristic parameters of spectral curve and chlorophyll content for rice].

It is of significance to monitor chlorophyll content with hyperspectral data for crop growth diagnosis in field. In the study, with the point of view that spectral curve shapes display "tall, low, fat and thin" morphological changes, we proposed some new characteristic parameters from spectral curve such as the ascensive or degressive velocities of segments composing peak or valley shapes in spectral curve, and angles formed by the lines fitting the segments of two sides of peak or valley curves, and used the normalized spectra to analyze correlation between these parameters and rice chlorophyll content. The result shows that (1) there is a good negative correlation between rice chlorophyll content and normalized reflectance spectra from 520-740 nm; (2) characteristic parameters from green peak region of spectral curve display better correlation with rice chlorophyll content, which makes it possible to utilize the parameters to monitor crop chlorophyll content, and will provide new ideas and methods for carrying out crop growth diagnosis with hyperspectral data.

Baculovirus surface display of E envelope glycoprotein of Japanese encephalitis virus and its immunogenicity of the displayed proteins in mouse and swine models.

Japanese encephalitis virus (JEV), an important pathogen in humans and animals, is capable of causing febrile syndrome, encephalitis and death. The E glycoprotein of JEV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. In this work, we have succeeded in construction of one recombinant baculovirus BacSC-E expressing His6-tagged E with the baculovirus envelope protein gp64 TM and CTD. After infection, E was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E glycoprotein was successfully displayed on the viral surface. Vaccination of mouse and swine with recombinant baculovirus BacSC-E successfully induced neutralizing antibody response and protective immunity toward a lethal challenge of the JEV. Taken all findings together, our results indicate that the recombinant baculovirus BacSC-E can be a potential vaccine against JEV infections. This finding provides valuable information for establishing subunit vaccines for JEV antigenic complex viruses. This is a fresh research demonstrating the potential of E-pseudotyped baculovirus as a JEV vaccine.

Development of ELISA kits for antibodies against avian reovirus using the sigmaC and sigmaB proteins expressed in the methyltropic yeast Pichia pastoris.

Both the sigmaC and sigmaB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigmaC, 1071-1 sigmaC, S1133 sigmaB, and S1133 sigmaC-sigmaB fusion genes of ARV were cloned into a secreted vector pPICZalphaA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigmaC, sigmaB, and sigmaC-sigmaB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1sigmaC and S1133 sigmaB and S1133 sigmaC-sigmaB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1sigmaC/S1133 sigmaB and S1133 sigmaC-sigmaB ELISA kits were 0.24 and 0.12, respectively. Forty-four serum neutralization test-positive and twenty-eight serum neutralization-negative samples from vaccinated and commercial farm chickens were tested by the new ELISA kits and by the conventional ELISA. The new ELISA kits have higher positive rates than the conventional ELISA. The results revealed that the correlation rates for the serum neutralization titer and the absorbance values with the new ELISA kits and the conventional ELISA were 100% and 95.8%, respectively.

Baculovirus surface display of NS3 nonstructural protein of classical swine fever virus.

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. NS3 proteins of CSFV, which include serine protease and RNA helicase/nucleotide triphosphatase (NTPase) activities, are multifunctional proteins involved in polyprotein processing and viral replication. Previous reports showed that NS3 protein can induce apoptosis in host cells that present cytopathic effects (CPE). Baculovirus/insect cell systems are used widely for recombinant protein production. In this study, one recombinant baculovirus BacSC-NS3 expressing histidine-tagged NS3 with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 of baculovirus was constructed. After infection, NS3 was expressed and anchored to the plasma membrane of Sf-9 cells, as demonstrated by Western blot assay and confocal microscopy. Immunogold electron microscopy demonstrated that the NS3 glycoprotein successfully displayed on the baculoviral envelope. Animal vaccine tests showed that recombinant baculovirus BacSC-NS3 elicited significantly higher NS3 antibody titers in the treated mouse models than the control group. This report demonstrated the potential of NS3-pseudotyped baculovirus expression of NS3 protein successfully.

Baculovirus surface display of E(rns) envelope glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) causes significant losses in the pig industry in many countries. E(rns) is an envelope glycoprotein of CSFV which is known to induce virus-neutralizing antibodies and protective immunity in the natural host. In this study, one recombinant baculoviruses BacSC-E(rns) expressing histidine-tagged E(rns) with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64 was constructed and its immunizing efficacy was evaluated in a mouse model. After infection, E(rns) was expressed and anchored on the plasma membrane of Sf-9 cells, as demonstrated by Western-blot and confocal microscopy. Immunogold electron microscopy demonstrated that the E(rns) glycoprotein was successfully displayed on the baculoviral envelope. Vaccine tests in animals showed that BacSC-E(rns) elicited significantly higher E(rns) antibody titers in the immunized mouse models than the control group. This demonstrates that the BacSC-E(rns) vaccine can be used potentially against CSFV infections. This is the first report demonstrating the potential of E(rns)-pseudotyped baculovirus as a CSFV vaccine.