Specific antibody responses
To determine the protective immune responses induced by the heterologous or homologous prime-boost strategies, neutralizing antibodies were measured by a neutralization assay at 3, 10, 17, 24, 31, 38, 48 and 60 dpi. The neutralizing antibody levels of the ads/ads and SE/SE groups gradually increased and reached a peak at 38 dpi and 48 dpi with 5.7 log2-fold and 6.2 log2-fold increases, respectively (Fig. 2A). For the SE/ads group, the level of neutralizing antibody reached a peak at 38 dpi, with 6.9 log2-fold, and maintained a high level after the peak. The peak of the SE/ads group was 2.4 and 1.4 times higher than those of the ads/ads and SE/SE groups, respectively. The overall level of neutralizing antibodies in the SE/ads group was higher than those of the ads/ads and SE/SE groups. For the WF/WF group, the levels of neutralizing antibodies reached a peak at 31 dpi, with 6.4 log2-fold, but decreased quickly after the peak. There were no neutralizing antibody responses in the serum of ducks from the PBS/PBS group (Fig. 2A).
A high level of specific antibodies against the E protein in the SE/ads group was observed, which showed a 5.6-fold higher level at 38 dpi compared with that in the PBS/PBS group (Fig. 2B). The antibodies in the SE/ads group remained at high levels from 38 to 60 dpi and were higher than those in the ads/ads and SE/SE groups from 24 to 60 dpi. For the WF/WF group, the antibody titre reached a peak at 31 dpi, which was 4.93-fold higher than that in the PBS/PBS group, but decreased quickly after the peak and was even lower than that in the SE/ads group after 31dpi (Fig. 2B).
The results herein showed that the heterologous prime-boost strategy could efficiently induce the production of higher titres of antibodies in ducks than the homologous prime-boost strategies.
Cytokines induced by the different immune strategies after immunization
To further characterize the induced immune response, quantitative RT-PCR was used to measure the cytokines (IFN-γ and IL-4) in the spleen. The expression level of IFN-γ in the ads/ads and SE/SE groups reached a peak that was 10.7-fold and 4.7-fold higher at 38 dpi and 31 dpi, respectively. For the SE/ads group, the expression level of IFN-γ increased quickly and exceeded that in the other three groups at 31 dpi. Then, it reached its peak at 48 dpi, which was 16.4-fold higher and maintained a high level of IFN-γ from 38 to 60 dpi. The expression level of IFN-γ in the WF/WF group, which reached a peak at 31 dpi with a 11.1-fold increase, was generally higher than that in the ads/ads and SE/SE groups, but lower than that in the SE/ads group after 31 dpi (Fig. 3A).
For IL-4, the expression levels of IL-4 in the ads/ads and SE/SE groups both reached peaks of 11.5-fold and 16.8-fold higher at 38 dpi, and the expression levels were lower than those in the SE/ads group from 17 to 31 dpi. Interestingly, the expression levels of IL-4 in the SE/ads group reached a peak at 31 dpi, which was 16.4-fold higher, but slowly decreased after the peak, and the levels were even lower than those in the SE/SE group after 31 dpi. For the WF/WF group, the expression level of IL-4 was generally lower than those in the SE/SE and SE/ads groups and lower than that in the ads/ads group after 31 dpi (Fig 3B).
Collectively, these results demonstrated that the heterologous prime-boost strategy resulted in stronger stimulation of IFN-γ (in the later stage) and IL-4 (in the early stage) after immunization than the homologous prime-boost strategies.
Protection of ducks against TMUV after challenge
To verify the clinical protection of ducks against TMUV infection, the immunized ducks were challenged with 1 ml of 105.1-fold ELD50 TMUV at 12 days after the boost vaccination (Table 1). All the ducks in the ads/ads, SE/ads and WF/WF groups survived. However, 10% and 30% of the ducks in the SE/SE and PBS/PBS groups died after challenge (Fig. 4A). Thus, the percent survival in the SE/SE and PBS/PBS groups was 90% (9/10) and 70% (7/10), respectively, while the percent survival in the ads/ads, SE/ads and WF/WF groups was 100% (10/10). To compare the protection against TMUV of these groups, the clinical signs were also recorded to evaluate the efficiency of these strategies. The ducks in the PBS/PBS group showed typical clinical signs after challenge, such as reluctance to move, head hung low, decreased appetite and neurological symptoms. However, slight clinical signs were also observed in the ads/ads and SE/SE groups, while no clinical signs were observed in the SE/ads and WF/WF groups. These results indicated that the heterologous prime-boost strategy provided better protection of the ducks against TMUV challenge than the homologous prime-boost strategies.
Viral loads in the tissues of ducks after challenge
The titres of TMUV were measured by quantitative RT-PCR based on the C gene of TMUV at 7 days after challenge in the heart, liver, spleen, kidney and brain. TMUV could be detected in all tissues from the five groups after challenge. The viral titres of the spleen were higher than those of the other tissues (heart, liver, kidney and brain) (Fig. 4B). The titres of TMUV in the SE/ads group were generally lower than those of the ads/ads and SE/SE groups in the heart, liver, spleen, kidney and brain. In the heart, the titres of TMUV in the SE/ads group were significantly lower than those in the ads/ads and SE/SE groups (P<0.01). In the liver, spleen and brain, the viral titres in the SE/ads group were significantly lower than those in the SE/SE group (P<0.01), but there was no significant difference from those in the ads/ads group. In the kidney, the titres of TMUV in the ads/ads, SE/SE and SE/ads groups were not significantly different from each other (Fig. 4B). In summary, the heterologous and homologous prime-boost regimens could both inhibit viral replication after challenge in ducks, but the heterologous prime-boost regimen could prevent viral replication better than the homologous prime-boost strategies.
Histopathological observation
The ducks were euthanized at 7 days after challenge, and their tissues (heart, liver, spleen, kidney and brain) were collected for histopathological observation. Histological observation of the different groups showed different histopathological changes. In the heart, slight myocardial fibre rupture and lymphocytic infiltration were found in the ads/ads and SE/SE groups (Fig. 5A and 5B), while no significant pathological damage was observed in the SE/ads and WF/WF groups (Fig. 5C and 5D). However, severe lesions in the PBS/PBS group were found with myocardial fibre rupture, oedema and lymphocytic infiltration (Fig. 5E).
In the liver, slight hepatocyte vacuolation was observed in the ads/ads group (Fig. 5F). Hepatocyte vacuolation and lymphocyte cell infiltration were observed in the SE/SE group (Fig. 5G), while the SE/ads and WF/WF groups had no obvious lesions (Fig. 5H and 5I). For the PBS/PBS group, the liver revealed severe hepatocyte vacuolation, hepatocyte necrosis and massive lymphocyte infiltration (Fig. 5J).
In the spleen, an obvious decrease in lymphocytes, an increase in reticulocytes and an unclear boundary between the red and white pulp were found in the ads/ads, SE/SE and PBS/PBS groups, while the other groups showed no significant lesions (Fig. 5K-5O).
In the kidney, no obvious pathological changes were observed in the ads/ads, SE/ads and WF/WF groups (Fig. 5P, 5R and 5S). The basement membranes of renal epithelial cells in the SE/SE and PBS/PBS groups were obviously detached (Fig. 5Q and 5T). Moreover, the kidney also experienced haemorrhage and necrosis in the epithelial cells of the PBS/PBS group (Fig. 5T).
In the brain, there were no obvious pathological changes in the ads/ads, SE/ads and WF/WF groups (Fig. 5U, 5W and 5X). The vascular sleeve phenomenon occurred in the brain tissues of the SE/SE group, while the PBS/PBS group showed both vascular sleeve and satellite phenomena (Fig. 5V and 5Y).
In summary, there were slight lesions in the ads/ads and SE/SE groups and no histological lesions in the SE/ads and WF/WF groups, and the most severe lesions were observed in the PBS/PBS group.