An Inactivated Novel Trivalent Vaccine Provides Complete Protection against FAdV-4 Causing Hepatitis-Hydropericardium Syndrome and FAdV-8b/-11 Causing Inclusion Body Hepatitis

Figures

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  Figure 1. Generation of recombinant rFAdV-4-fiber/8b + 11. (a) Strategy for generation of rFAdV-4-fiber/8b + 11. (b) Identification of rFAdV-4-fiber/8b + 11 by western blot.

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  Figure 2. The rFAdV-4-fiber/8b + 11 replicated efficiently in LMH cells. LMH cells were infected with rFAdV-4-fiber/8b + 11 and wild-type FAdV-4 at same dose (MOI = 0.001), respectively, and the TCID50 of each virus collected at the indicated time points was determined in three technical replicates and presented as the mean ± standard error of mean.

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  Figure 3. Inactivated rFAdV-4-fiber/8b + 11 vaccine induced specific immune responses against FAdV-4, FAdV-8b, and FAdV-11. The antibody responses against FAdV-4, FAdV-8b, and FAdV-11 was evaluated by an indirect ELISA. The mean OD450 nm absorbance values and standard error of the mean are shown for each group. The dashed line represents the background level of detection.

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  Figure 4. Percent of survival for chickens challenged with virulent FAdV-4 (a), FAdV-8b (b), and FAdV-11 (c). Chickens from each group were intramuscularly challenged with either 2 × 105 TCID50 of FAdV-4 or 2 × 106 TCID50 of FAdV-8b or FAdV-11, respectively. The challenged chickens were monitored daily for 7 days and survival rates were calculated.

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  Figure 5. The representative gross lesion in the heart, liver, and kidney from chickens in each group after challenging with FAdV-4 (a), FAdV-8b (b), and FAdV-11 (c). (a) Chickens in the unvaccinated and FAdV-4 challenge group showed accumulation of clear straw-colored fluid in the pericardial sac, swollen and discolored liver, and enlarged kidneys with distended tubules. (b) and (c) Chickens in the FAdV-8b or FAdV-11 challenge control group exhibited IBH-indicative gross lesions, characterized by swollen livers with small white focal necrosis and petechial hemorrhages.

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  Figure 6. Representative histopathological lesions in tissues from chickens in each group after challenging with FAdV-4, FAdV-8b, and FAdV-11. (a) Histopathological lesions including widened myocardial cell gap and myocardial fiber rupture; severe degeneration and necrosis of hepatocytes; degeneration and necrosis of renal tubular epithelium; mucosal epithelial cell nuclear fragmentation and necrosis in duodenums; and severe depletion and necrosis of lymphocytes in the bursa of Fabricius were observed in the chickens from the unvaccinated and FAdV-4 challenge group. (b) Compared with the massive severe degeneration and necrosis of hepatocytes caused by FAdV-4, the primary histopathological lesions presented by the unvaccinated and FAdV-8b or FAdV-11 challenge groups included multifocal degeneration and necrosis of hepatocytes and mononuclear cell infiltration. However, chickens from the inactivated rFAdV4-fiber/8b + 11 vaccinated group did not present histopathological lesions of HHS and IBH mentioned above (HE staining, original magnification ×400).

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  Figure 7. Viral loads in tissues from chickens in each group after challenging with FAdV-4 (a), FAdV-8b (b), and FAdV-11 (c). A SYBR Green qRT-PCR was used to determine the viral loads in tissues. The viral loads were calculated as copy numbers/milligram tissue and presented as the means ± standard error of the mean (∗, ∗∗, and ∗∗∗ indicate P < 0.05, 0.01, and 0.001, respectively). The dashed line represents the background level of detection.