Pathological Evaluation of Porcine Circovirus 2d (PCV2d) Strain and Comparative Evaluation of PCV2d and PCV2b Inactivated Vaccines against PCV2d Infection in a Specific Pathogen-Free (SPF) Yucatan Miniature Pig Model

Abstract

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot^® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.

Keywords: PCV2b; PCV2d; Yucatan miniature pig; cross-protection; inactivated vaccine; porcine circovirus type 2 (PCV2).

Figure 1

Maximum likelihood (ML) phylogenetic tree of the PCV2 ORF2 gene. The tree was generated using MEGA X software with 1000 bootstrap replicates. The PCV2d isolate used in this study (CBNU0324) is indicated with a red square symbol.

Figure 2

Growth performance of mock and PCV2d infection groups in the experimental infection/pathogenicity study (Experiment 1). The average daily weight gain (ADWG) was calculated during experimental periods: overall PCV2d infection (0–21 dpi, 21 days) (A), and each week after PCV2d infection (0–7 dpi, 7–14 dpi, and 14–21 dpi) (B). All data are presented as group means ± SEM. Statistically significant differences are indicated at p < 0.001 (***), and p < 0.0001 (****).

Figure 3

PCV2 genome copies and PCV2-specific IgG levels in the experimental infection/pathogenicity study (Experiment 1). Quantitative PCR analysis of PCV2 genome copies in the PCV2d infection group (A), and the levels of PCV2-specific antibodies in the mock and PCV2d infection groups (B). All data are presented as group means ± SEM.

Figure 4

PCV2 genome copies, histological and immunohistochemical findings from PCV2d infection group in the experimental infection/pathogenicity study (Experiment 1). The levels of PCV2 genome copies in multiple organs obtained from the PCV2d infection group (A), and representative images of histological findings by H&E staining ((B), magnification: ×100) or immunohistochemistry staining ((C), magnification: ×200) within tissue samples obtained from the PCV2d infection group. The data of PCV2 genome copies are presented as group means ± SEM. Immunolabeling of PCV2 was observed as a dark brown signal.

Figure 5

Growth performance of vaccinated and nonvaccinated pigs in the vaccine efficacy study (Experiment 2). The average daily weight gain (ADWG) was calculated during three experimental periods: vaccination to PCV2d challenge (0–21 dpv, 21 days) (A), PCV2d challenge to necropsy (0–21 dpc, 21 days) (B), and each week after PCV2d challenge (0–7 dpc, 7–14 dpc, and 14–21 dpc) (C). All data are presented as group means ± SEM. Statistically significant differences are indicated at p < 0.0001 (****).

Figure 6

Antibody responses in pigs after immunization with different PCV2 vaccines in the vaccine efficacy study (Experiment 2). The levels of PCV2-specific IgG antibodies (A) and neutralizing antibodies (B) were detected at the indicated timepoints. All data are presented as group means ± SEM. Statistically significant differences between Non-Vac and vaccinated groups within the timepoint are indicated at p < 0.0001 (****).

Figure 7

Quantitative PCR analysis of PCV2 genome copies in vaccinated and nonvaccinated pig serum (A), nasal swab samples (B), and tissue samples (C) in the vaccine efficacy study (Experiment 2). The viral DNA copies in the serum and nasal swab samples were detected by quantitative PCR at the indicated timepoints, and those in lung, lymph node, and spleen samples were detected at the time of necropsy. All data are presented as group means ± SEM. Statistically significant differences (p < 0.001, Tukey’s multiple-comparison test) between Non-Vac and vaccinated groups are indicated at p < 0.001 (***), and p < 0.0001 (****).

Figure 8

Representative images of immunohistochemistry assessment of PCV2 antigen in lung, lymph node, and spleen from PCV2b-Vac, PCV2d-Vac, and Non-Vac groups in the vaccine efficacy study (Experiment 2). Immunolabeling of PCV2 was observed as a dark brown signal. Magnification: ×400.