Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis.

OBJECTIVE: The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. METHODS: The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. RESULTS: The optimal antigen coating concentration was 2 µg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. CONCLUSIONS: The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

Phage display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential.

The spike (S) protein of porcine transmissible gastroenteritis virus (TGEV) is located within the viral envelope and is the only structural protein that possesses epitopes capable of inducing virus-neutralizing antibodies. Among the four N-terminal antigenic sites A, B, C, and D, site A and to a lesser extent site D (S-AD) induce key neutralizing antibodies. Recently, we expressed S-AD (rS-AD) in recombinant form. In the current study, we used the rS-AD as an immobilized target to identify peptides from a phage-display library with application for diagnosis. Among the 9 phages selected that specifically bound to rS-AD, the phage bearing the peptide TLNMHLFPFHTG bound with the highest affinity and was subsequently used to develop a phage-based ELISA for TGEV. When compared with conventional antibody-based ELISA, phage-mediated ELISA was more sensitive; however, it did not perform better than semi-quantitative RT-PCR, though phage-mediated ELISA was quicker and easier to set up.

Generation of a monoclonal antibody to S1 protein of porcine epidemic diarrhea virus.

Gene encoding the N-terminal half of spike protein (S1) of porcine epidemic diarrhea virus (PEDV) was cloned and expressed as a recombinant protein in Escherichia coli BL21 (DE3). Then, female BALB/c mice were immunized with the purified recombinant S1 protein (rS1), and a monoclonal antibody (MAb designated as 5E12) against the rS1 protein was achieved by hybridoma technique. MAb 5E12 not only reacted with rS1 protein indirect ELISA and Western blot, but also recognized PEDV transiently expressed in Vero E6 cells in indirect immunofluorescence examinations. This work suggests that 5E12 would be a useful tool as a specific diagnostic reagent for detecting PEDV S protein.

Development of porcine rotavirus vp6 protein based ELISA for differentiation of this virus and other viruses.

BACKGROUND: The context and purpose of the study included 1) bacterial expression of viral protein 6 (VP6) of porcine rotavirus (PRV) and generation of rabbit polyclonal antiserum to the VP6 protein; 3) establishment of a discrimination ELISA to distinguish PRV from a panel of other porcine viruses. RESULTS: The VP6 gene of PRV isolate DN30209 amplified by reverse transcription-PCR was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. Sequence comparison and phylogenetic analysis indicated that PRV DN30209 may belong to group A of rotavirus. Bacterially expressed VP6 was expressed in E.coli and anti-VP6 antibody was capable of distinguishing PRV from Porcine transmissible gastroenteritis virus, Porcine epidemic diarrhea virus, Porcine circovirus type II, Porcine reproductive and respiratory syndrome virus, Porcine pseudorabies virus and Porcine parvovirus. CONCLUSIONS: PRV VP6 expressed in E. coli can be used to generate antibodies in rabbit; anti-VP6 serum antibody can be used as good diagnostic reagents for detection of PRV.

Incidence rates and deaths of tuberculosis in HIV-negative patients in the United States and Germany as analyzed by new predictive model for infection.

Incidence and mortality due to tuberculosis (TB) have been decreasing worldwide. Given that TB is a cosmopolitan disease, proper surveillance and evaluation are critical for controlling dissemination. Herein, mathematical modeling was performed in order to: 1) demonstrate a correlation between the incidence of TB in HIV-free patients in the US and Germany, and their corresponding mortality rates; 2) show the utility of the newly developed D-R algorithm for analyzing and predicting the incidence of TB in both countries; and 3) inform us on population death rates due to TB in HIV-negative patients. Using data published by the World Health Organization between 1990 and 2009, the relationship between incidence and mortality that could not be ascribed to HIV infection was evaluated. Using linear, quadratic and cubic curves, we found that a cubic function provided the best fit with the data in both the US (Y = 2.3588+2.2459X+61.1639X(2)-60.104X(3)) and Germany (Y = 1.9271+9.4967X+18.3824X(2)-10.350X(3)) where the correlation coefficient (R) between incidence and mortality was 0.995 and 0.993, respectively. Second, we demonstrated that fitted curves using the D-R model were equal to or better than those generated using the GM(1,1) algorithm as exemplified in the relative values for Sum of Squares of Error, Relative Standard Error, Mean Absolute Deviation, Average Relative Error, and Mean Absolute Percentage Error. Finally, future trends using both the D-R and the classic GM(1,1) models predicted a continued decline in infection and mortality rates of TB in HIV-negative patients rates extending to 2015 assuming no changes to diagnosis or treatment regimens are enacted.

Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA constructs in the eukaryotic expression plasmid pVAX1; one encoding the S protein [pVAX1-(PEDV-S)], the second encoding the N-terminal fragment (S1) [pVAX1-(PEDV-S1)] containing potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18) [pVAX1-(IL-18)]. Immunofluorescence assays in BHK-21 cells demonstrated successful protein expression from all 3 constructs. Kunming mice were injected separately with each of these constructs or with a pVAX1-(PEDV-S1)/pVAX1-(IL-18) combination, an attenuated PEDV vaccine, or vector only control. Animals were examined for T lymphocyte proliferation, anti-PEDV antibodies, IFN-γ and IL-4 protein levels, and cytotoxic T cell function in mouse peripheral blood and spleen. In all cases, results showed that pVAX1-(PEDV-S) and the combination of pVAX1-(PEDV-S1) with pVAX1-(IL-18) induced the strongest responses; however, pIL-18 had no adjuvant effects when given in combination with pVAX1-(PEDV-S1).

[A herbalogical study on traditional Mongolian medicine "zhanba"].

By herbalogical study and investigation, "Zhanba" used by Mongolia doctors mainly contains 7 species from 3 genera of 1 family, but "Zhanba" in Inner Mongolia Standard of Medicinal Materials only contains 3 species which are Althaea rosea, Malva sylvestris L. var. mauritiana and M. verticillata.