Development of an Effective Single-Dose PCV2/CSFV Bivalent Subunit Vaccine Against Classical Swine Fever Virus and Porcine Circovirus Type 2

Figures

• 

  Figure 1. The schematic representation of the animal experimental design. The bleeding times were represented by three symbols: ●, vaccination trial: vaccinated pigs (Groups A and C) and non-vaccinated pigs (Groups C and D) were immunized with the PCV2/CSFV bivalent vaccine or saline, respectively, at week 0 (1st vaccination); ▲, CSFV challenge trial: pigs in Groups A and B were challenged with the CSFV ALD strain (105.44 FAID50/total dose per pig) at the beginning of week 5. Blood samples were collected at days 0, 4, 7, 10, and 14 post-challenge, and pigs were sacrificed on day 14 post-challenge (week 7); ■, PCV2 challenge trial: pigs in Groups C and D were challenged with the PCV2d THF0601-7 strain (106.90 TCID50/total dose per pig) at the beginning of week 6. Blood samples were collected at weeks 0, 2, 3, 4, 5, 6, and 7 post-challenge, and pigs were sacrificed at week 7 post-challenge (week 13).

• 

  Figure 2. The expression of CSFV-E2 and PCV2-ORF2 proteins from ExpiCHOTM stable cell line. CSFV-E2 (A) and PCV2-ORF2 (B) proteins were expressed by Western blot. CSFV-E2 protein reduced with or without reagent β-ME (Lane 1 and Lane 2, respectively). Monoclonal antibody (mAb) WH303 and PCV2 capsid polyclonal antibody were used for the Western blot, respectively. Lane M indicates protein markers.

• 

  Figure 3. The effect of bivalent vaccine after vaccination in response to CSV or PCV2 sera antibody titers. Sera samples from all pigs were collected and evaluated for CSFV E2-specific Ab (A) and PCV2 ELISA Ab (B) responses, respectively. Results are values ≥ 40% and ≥ 0.5 that are considered positive by the IDEXX CSFV Ab test (blocking%) and BioChek PCV2 Ab test (S/P ratio), respectively. Data with different letters (a,b) indicate that the groups are significantly (p < 0.05) different from each other at the same time point.

• 

  Figure 4. The evaluation of the clinical symptoms of pigs before (day 0) and after the CSFV challenge. Rectal temperatures (A) and clinical scores (B) were determined in pigs. All pigs were challenged with CSFV (105.44 FAID50/total dose per pig) four weeks after vaccination and sacrificed on day 14 post-challenge. Data were expressed as mean ± SEM of rectal temperatures. In addition, the non-parametric Mann–Whitney U test was used to analyze clinical scores, and data were expressed as the median and 95% confidence intervals. Data with different letters (a,b) indicate that the groups are significantly (p < 0.05) different from each other at the same time point.

• 

  Figure 5. Serological responses in pigs challenged with CSFV or PCV2. Vaccinated (Groups A and C) and non-vaccinated (Groups B and D) pigs received a CSF/PCV bivalent vaccine or saline on day −28 and were challenged on day 0. Sera were analyzed for CSFV E2-specific (A), PCV2 ELISA (B,C), and CSFV-neutralizing (D) antibodies. Results are values ≥ 40% and ≥ 0.5 that are considered positive by the IDEXX CSFV Ab test (blocking%) and BioChek PCV2 Ab test (S/P ratio), respectively. Data are presented as mean ± SEM. Different letters (a,b) indicate significant differences (p < 0.05) at the same time point.

• 

  Figure 6. Viral loads in tissue were determined after two challenge trials. Tissues in pigs were collected and examined for viral loads of CSFV (A) or PCV2 (B) after euthanasia. Data in CSFV challenge trial were expressed as mean ± SEM of log10 TCID50/g, and in PCV2 trial as mean ± SEM of log10 copies/g. Data with different letters (a,b) indicate that the groups are significantly (p < 0.05) different from each other at the same time point.