A prospective CSFV-PCV2 bivalent vaccine effectively protects against classical swine fever virus and porcine circovirus type 2 dual challenge and prevents horizontal transmission

Figures

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  Figure 1. CSFV-PCV2 bivalent vaccine provokes immune responses in a mice model. ICR mice were intraperitoneally immunized (0.5 mL/dose) with CSFV-PCV2 bivalent vaccine (n = 5) or placebo (n = 5) at 6 and 8 weeks of age. Serum samples were collected at 12 weeks of age to evaluate CSFV-specific (A) or PCV2-specific (B) antibody levels. Splenocytes were isolated at 14 weeks of age to evaluate the number of CSFV-specific (C) and PCV2-specific (D) IFN-γ-secreting cells by ELISpot assay. Data are presented in a box plot, and each spot indicates an individual mouse. The Kruskal–Wallis test was used for statistical analysis, and differences were considered statistically significant at a p value < 0.05.

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  Figure 2. CSFV-PCV2 bivalent vaccine provokes antigen-specific cellular immune response in SPF pigs. SPF pigs were immunized with two doses of CSFV-PCV2 bivalent vaccine (Group A, n = 4) or placebo (Group C, n = 4) at 6 and 8 weeks of age. PBMCs were isolated and stimulated with the viruses (CSFV LPC strain and PCV2) and subunit proteins (CSF-E2 and PCV2 capsid protein). The antigen-specific IFN-γ secreting cells for 1 MOI LPC virus (A), 10 µg of CSF-E2 subunit protein (B), 1 MOI PCV2 virus (C), and 10 µg of PCV2 capsid protein (D) were calculated. Data are presented in a box plot, and each spot indicates an individual mouse. The Kruskal–Wallis test was used for statistical analysis, and differences were considered statistically significant at a p value < 0.05.

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  Figure 3. Bivalent vaccine protected immunized pigs against CSFV and PCV2 virus challenge and prevented horizontal transmission. SPF pigs immunized with CSFV-PCV2 bivalent vaccine immunization (Group A, n = 4) or placebo (Group B, n = 4) were challenged with 1.5 × 106 TCID50 of CSFV (ALD strain) and 1 × 105 TCID50 of PCV2 (CYC08 strain) at 12 weeks of age. Three days after challenge, the sentinel pigs (Group C, n = 4) were cohabitated with Group A to monitor virus horizontal transmission. Pigs in Group B were euthanized at 13 weeks of age (1 week post-challenge) due to severe clinical signs of infection and weakness, whereas pigs in Groups A and C were euthanized at 15 weeks of age. The clinical signs (A), body temperature (B), and average daily weight gain (C) were monitored and recorded as indicators for virus-induced disease. Serum samples were utilized for virus screening (D, F) and dynamic antibody levels (E, G). Data are presented as the mean ± standard error of the mean.

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  Figure 4. Application of the CSFV-PCV2 bivalent vaccine to conventional pigs. Conventional pigs were divided into two groups and immunized with one dose of CSFV-PCV2 bivalent vaccine (Group D, n = 5) and placebo (Group E, n = 5) at 4 weeks of age. All pigs were challenged with 1 × 105 TCID50 of PCV2 at 8 weeks of age. The CSFV-specific antibody response was evaluated by neutralization assay (A) and ELISA (B). The serum PCV2-specific antibody level and PCV2 viral load were monitored by ELISA (C) and real-time PCR (D). All pigs were sacrificed at 12 weeks of age. The peripheral lymph nodes were subjected to viral load detection by real-time PCR (E) and IHC analysis (F). Data are presented as the mean ± standard error of the mean, and the Kruskal–Wallis test was used for statistical analysis. Differences were considered statistically significant at a p value < 0.05.

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  Figure 5. Analysis of the histopathological changes in the peripheral lymph nodes after PCV2 challenge. The lymph nodes from vaccinated (Group D) and placebo-immunized (Group E) pigs, including the submandibular, hilar, mesenteric, and inguinal lymph nodes were collected at 4 weeks after PCV2 challenge and were processed for H&E and IHC staining for the PCV2 antigen. (left: H&E staining, right: IHC staining specific for PCV2 capsid protein).