Journal: PLoS ONE

Article Title: Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

doi: 10.1371/journal.pone.0098048

Figure Lengend Snippet: Oligonucleotide primers and RT-PCR conditions for the analysis of mRNA levels.

Article Snippet: For the quantitative determination of pig C3 protein concentration in serum samples, a commercially available sandwich ELISA was used (Pig Complement C3 ELISA kit, CUSABIO, Wuhan, China).

Techniques:

Journal: PLoS ONE

Article Title: Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

doi: 10.1371/journal.pone.0098048

Figure Lengend Snippet: (A) Identification by Western blot and mass spectrometry of mycobacterial proteins recognized by antibodies in IV vaccinated wild boar. Identified proteins are indicated with arrows in Coomassie-blue stained 12% SDS-polyacrylamide gel (left panel) and Western blot with pooled sera from vaccinated and control animals at T0 and T2 (right panel) with 10 µg IV proteins. MW, molecular weight markers. (B) Expression of recombinant proteins in E. coli . Ten µg of purified proteins were loaded per well in a 12% SDS-polyacrylamide gel. Recombinant proteins are indicated with arrows. MW, molecular weight markers. (C) Western blot analysis of recombinant mycobacterial proteins using pooled sera from IV vaccinated and control animals at T0, T1 and T2. Ten µg of purified proteins were loaded per well in a 12% SDS-polyacrylamide gel. The position of the recombinant proteins is indicated with arrows. (D) Antibody levels against mycobacterial proteins were determined by ELISA in serum samples from vaccinated and control animals and O.D. 450 nm values (Ave+S.D.) were compared at each time point between vaccinated and control groups by Student’s t-test with unequal variance (*P≤0.05). (E, F) Correlation analysis between antibody levels against NADPAD and MPB83 mycobacterial proteins in vaccinated and control animals and (E) lesion score or (F) culture score at T2 (N = 15).

Article Snippet: For the quantitative determination of pig C3 protein concentration in serum samples, a commercially available sandwich ELISA was used (Pig Complement C3 ELISA kit, CUSABIO, Wuhan, China).

Techniques: Western Blot, Mass Spectrometry, Staining, Control, Molecular Weight, Expressing, Recombinant, Purification, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

doi: 10.1371/journal.pone.0098048

Figure Lengend Snippet: (A) Comparison of PBMC mRNA levels between vaccinated (N = 7) and control (N = 8) animals. (B) Comparison of PBMC mRNA levels between control animals with lower (N = 5) and higher (N = 3) tuberculous lesion score. The mRNA levels of selected genes were analyzed by real-time RT-PCR in PBMC of vaccinated and control wild boar collected at T0 (day 1, before vaccination), T1 (day 126, before challenge) and T2 (day 255 at necropsy). The mRNA levels were normalized against S. scrofa cyclophilin, β-actin and GAPHD and normalized Ct values were represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05). (C) Serum C3 protein levels (µg/ml) were determined by ELISA in vaccinated and control wild boar at T0, T1 and T2 and compared between groups by Student’s t-test with unequal variance (P>0.05). (D) The difference in serum C3 protein levels (µg/ml) determined by ELISA in vaccinated and control wild boar was calculated between T2 and T1 and represented as Ave±S.D. (E) Serum cytokine protein levels were determined using the Quantibody porcine cytokine array in vaccinated (N = 7) and control (N = 8) wild boar at T0, T1 and T2, represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05).

Article Snippet: For the quantitative determination of pig C3 protein concentration in serum samples, a commercially available sandwich ELISA was used (Pig Complement C3 ELISA kit, CUSABIO, Wuhan, China).

Techniques: Comparison, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

doi: 10.1371/journal.pone.0098048

Figure Lengend Snippet: (A) Total RNA (4 µg) extracted from the IV or IV (2 µl) were subjected to different treatments and analyzed by agarose gel electrophoresis after RT-PCR for the amplification of Mycobacterium spp. 16S rRNA. (B) Total RNA extracted from the IV was treated with DNase I and/or RNase and used for cDNA synthesis using random primers 16S rRNA PCR. (C) Radial un-rooted tree of the 16S rRNA phylogenetic analysis using Neighbor Joining. Mycobacterium species and sequence Genbank accession numbers are shown. (D) Alignment of 16S rRNA sequences from the same mycobacteria used in the phylogenetic analysis to show characteristic single nucleotide polymorphisms in the vaccination isolate ( M. bovis (IV)). (E) C3 mRNA levels in the oral mucosa and PBMC at T2. (F) Serum C3 protein levels in pigs vaccinated with the IV and IV-DNA/RNA determined by ELISA at T0, T1 and T2. The mRNA levels were normalized against S. scrofa cyclophilin, β-actin and GAPHD. Normalized Ct values and protein levels (µg/ml) were represented as Ave+S.D. and compared between groups by Student’s t-test with unequal variance (*P≤0.05).

Article Snippet: For the quantitative determination of pig C3 protein concentration in serum samples, a commercially available sandwich ELISA was used (Pig Complement C3 ELISA kit, CUSABIO, Wuhan, China).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, cDNA Synthesis, Sequencing, Enzyme-linked Immunosorbent Assay