Specific antibody responses
In order to determine the protective immune responses induced by the heterologous or homologous prime-boost strategies, the neutralizing antibodies were measured by neutralizing assay at 3, 10, 17, 24, 31, 38, 48 and 60 dpi. The neutralizing antibody levels of ads/ads and SE/SE group gradually increased and reached to the peak at 38 dpi and 48 dpi with 5.7 log2-folds and 6.2 log 2-folds (Fig. 2A). As for SE/ads group, the level of neutralizing antibody reached to the peak at 38 dpi with 6.9 log2-folds, and maintained a high level after the peak. The peak of SE/ads group was 2.4 and 1.4 times higher than those of ads/ads and SE/SE groups, respectively. And the overall level of neutralizing antibodies in SE/ads was higher than that of the ads/ads and SE/SE groups. As for the WF/WF group, the levels of neutralizing antibodies reached to the peak at 31 dpi with 6.4 log2-folds, but decreased quickly after the peak. There were no neutralizing antibody responses in the serum of ducks from PBS/PBS group (Fig. 2A).
A high level of specific antibodies against E protein in SE/ads was observed, which showed a 5.6-fold higher level at 38 dpi compared with the PBS/PBS group (Fig. 2B). And the antibodies in SE/ads group remained at high levels from 38 to 60 dpi and were higher than that in ads/ads and SE/SE from 24 to 60 dpi. As for the WF/WF, the titers of antibodies reached to the peak at 31 dpi with 4.93-fold higher than that in PBS/PBS group, but decreased quickly after the peak, and even lower than that in SE/ads after 31dpi (Fig. 2B).
The results herein suggested that the heterologous prime-boost strategy could efficiently induce the production of higher titers of antibodies in ducks than the homologous prime-boost strategies.
Cytokines induced by the difference immune strategies after immunization
To further characterize the induced immune response, the quantitative RT-PCR was used to measure the cytokines (IFN-γ and IL-4) in the spleen. The expression level of IFN-γ in ads/ads and SE/SE reached to the peak with 10.7-fold and 4.7-fold higher at 38 dpi and 31dpi. As for the SE/ads, the expression level of IFN-γ increased quickly and exceeded the other three groups after 31dpi. Then it reached to the peak at 48 dpi with 16.4-fold higher and maintained a high level of IFN-γ from 38 to 60 dpi. The expression level of IFN-γ in WF/WF, which reached to the peak at 31 dpi with 11.1-fold higher, was generally higher than that in ads/ads and SE/SE, and lower than that in SE/ads after 31 dpi (Fig. 3A).
As for the IL-4, the expression levels of IL-4 in ads/ads and SE/SE both reached to the peak with 11.5-fold and 16.8-fold higher at 38 dpi, and the expression levels were lower than that in SE/ads from 17 to 31dpi. Interestingly, the expression levels of IL-4 in SE/ads reached to the peak at 31 dpi with 16.4-fold higher, but slowly decreased after the peak, and the levels was even lower than that in SE/SE after 31 dpi. As for WF/WF, the expression level of IL-4 was generally higher than that in SE/SE and SE/ads groups, but lower than that in ads/ads after 31 dpi (Fig. 3B).
Collectively, these results demonstrated that the heterologous prime-boost strategy resulted in strong stimulation of IFN-γ (in the later stage) and IL-4 (in the early stage) after immunization than the homologous prime-boost strategies, which indicated that the heterologous prime-boost strategy could induce better cellular immune response against TMUV.
Protection of ducks against TMUV after challenging
To verify the clinical protection of ducks against TMUV infection, the immunized ducks were challenged with 1 ml 105.1-fold ELD50 TMUV at 12 days after the boost vaccination (Table 1). All the ducks in ads/ads, SE/ads and WF/WF groups were survived. However, 10% and 30% ducks in SE/SE and PBS/PBS groups died after challenging (Fig. 4A). Thus, the survival ratio in SE/SE and PBS/PBS were 90% and 70%, while the survival ratio in ads/ads, SE/ads and WF/WF were 100%. In order 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 PBS/PBS showed typical clinical signs after challenging, such as depression, decreasing appetite and neurological symptoms. However, slight clinical signs were also observed in ads/ads and SE/SE groups while no clinical signs were observed in SE/ads and WF/WF groups. These results indicated that the heterologous prime-boost strategy was with a better protection of the ducks against the challenging of TMUV than that of the homologous prime-boost strategies.
Viral loads in the tissues of ducks after challenging
The titers of TMUV were measured by quantitative RT-PCR based on the C gene of TMUV at 7 days after challenging in heart, liver, spleen, kidney and brain. The TMUV could be detected in all tissues from the five groups after challenging. The viral titers of the spleen were higher than that of the other tissues (heart, liver, kidney and brain) (Fig. 4B). The titers of TMUV in SE/ads was generally lower than that of ads/ads and SE/SE groups in heart, liver, spleen, kidney and brain. In heart, the titers of TMUV in SE/ads was significantly lower than that in ads/ads and SE/SE groups (P༜0.01). In liver, spleen and brain, the viral titers in SE/ads were significantly lower than that in SE/SE group (P༜0.01), but no significant difference with that in ads/ads groups. In kidney, the titers of TMUV in ads/ads, SE/SE and SE/ads had no significant difference with each other (Fig. 4B). To sum up, the heterologous and homologous prime-boost regimens could both inhibit the viral replication after challenging in ducks, but the heterologous prime-boost regimen could better prevent the viral replication than the homologous prime-boost strategies.
Histopathological Observation
The ducks were euthanized at 7 days after challenging, and their tissues (heart, liver, spleen, kidney and brain) were collected for histopathological observation. Histological observation with different groups showed different histopathological changes. In heart, slight myocardial fiber rupture and lymphocytic infiltration were found in the ads/ads and SE/SE groups (Fig. 5A and 5B), while no significant pathological damages were observed in SE/ads and WF/WF groups (Fig. 5C and 5D). But severe lesions in the PBS/PBS group were found with myocardial fiber rupture, edema and lymphocytic infiltration (Fig. 5E).
In liver, it showed slight hepatocyte vacuolation in ads/ads group (Fig. 5F). Hepatocyte vacuolation and lymphocyte cell infiltration were observed in SE/SE group (Fig. 5G), while the SE/ads and WF/WF groups had no obvious lesions (Fig. 5H and 5I). As for the PBS/PBS group, the liver revealed severe hepatocyte vacuolation, hepatocyte necrosis and massive lymphocyte infiltration (Fig. 5J).
In spleen, obvious decreasing lymphocytic, increasing reticulocyte and unclear boundary between red and white pulp were founded in ads/ads, SE/SE and PBS/PBS groups, while the other groups showed no significant lesions (Fig. 5K-5O).
In 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 detached obviously (Fig. 5Q and 5T). Moreover, the kidney also experienced bleeding and necrotic in the epithelial cells of the PBS/PBS group (Fig. 5T).
In brain, there were no obvious pathological changes in ads/ads, SE/ads and WF/WF groups (Fig. 5U, 5W and 5X) while observed. Vascular sleeve phenomenon occurred in the brain tissues of the SE/SE group while the PBS/PBS showed both vascular sleeve and satellite phenomenon (Fig. 5V and 5Y).
In summary, there were slight lesions in ads/ads and SE/SE groups, no microscopic lesions in SE/ads and WF/WF groups, but the most severe lesions in PBS/PBS group.