Table 1.
Oligonucleotide primers used in the research.
Fig 1.
Confirmation of PmpD-N protein expression in HVT vector by immunoblotting assay and indirect immunofluorescence.
(A) The PmpD-N expression in rHVT-pmpD-N was detected by immunoblot with C. psittaci strain 6BC-specific polyclonal antibodies. Lane M, pre-stained protein ladder; Lane 1, cell lysate post inoculation with rHVT-pmpD-N; Lane 2, cell lysate post inoculation with parental HVT. The black arrow indicates the approximately size of 43kDa. (B) Indirect immunofluorescence analysis of PmpD-N expression in CEF cells. CEF cells on glass coverslips were infected with rHVT-pmpD-N, then incubated with mouse anti-PmpD-N polyclonal antibody of C. psittaci and chicken anti-HVT polyclonal serum, and then reacted with goat anti-mouse IgG conjugated with Alexa Fluor 488 (green fluorescence, shown in the lower left panel) and goat anti-chicken IgY labelled with Alexa Fluor 568 (red fluorescence, shown in the lower right panel), respectively. Finally, cell nuclei were stained with DAPI (blue fluorescence, shown in the top right panel). The merged image is shown in the top left panel. The expression of the targeted protein is indicated by white arrows in top left panel. (C) Parental HVT control. CEF cells on glass coverslips were infected with parental HVT, and then the process of test and the panel meaning are the same as those shown in Fig 1B.
Fig 2.
One-step growth kinetics of rHVT-pmpD-N and parental HVT in vitro and in vivo.
(A) The virus growth was calculated as the fold increase at different time points compared with the 12th h post infection. The asterisk indicates significant differences of the growth kinetics between rHVT-pmpD-N and parental HVT (P<0.05). (B) The replication kinetics of parental HVT (n = 10) and rHVT-pmpD-N (n = 15) in vaccinated chickens was evaluated by real time PCR in PBLs. The HVT genome copies were quantified in 75 ng extracted DNA at different time point. The values were shown as means ± standard deviation.
Table 2.
Protective efficacy of rHVT-pmpD-N and parental HVT against challenge with very virulent MDV strain RB-1B.
Fig 3.
Sera from CEF control group (n = 10), parental HVT group (n = 10) and rHVT-pmpD-N group (n = 15) were analyzed by ELISA assay for anti-PmpD-N antibody using plates coated with PmpD-N as the antigens as described in material and method section. Results are expressed as absorbance at 450nm/630nm. A serum sample was considered positive when its OD value exceeded 0.045. The values were shown as means ± standard deviation. ** Indicates P < 0.01 when rHVT-pmpD-N & parental HVT group or CEF control group.
Fig 4.
Antigen-specific proliferation responses.
PBLs (n = 5 for each group) were obtained from immunized chickens on days 35, and lymphocytes were stimulated with 400 PFU of inactivated rHVT-pmpD-N. The results were examined as the stimulation index, calculated as the mean counts per minute (cpm) values of stimulated and non-stimulated wells. The values were shown as dot plots and means. a–cBars with the different superscripts indicate statistically significant difference among three groups.
Fig 5.
T lymphocyte subsets induced by immunization with rHVT-pmpD-N and parental HVT.
T cells were purified from the blood of immunized chickens (n = 5 for each group) on day 35. The values were shown as dot plots and means. (A) The proportion of T cell subsets in PBLs by flow cytometric analysis. a–bBars in the same T lymphocyte subsets with the different superscripts indicate significantly different among three groups. (B) The ratio of CD4+/CD8+ among three groups. a–bBars with the different superscripts indicate statistically significant difference among three groups.
Fig 6.
Lesion scores in chickens after challenge.
Gross lesions (n = 10 for CEF control group and parental HVT group, n = 15 for rHVT-pmpD-N group) in target organs were assessed, and higher scores represent more severe lesions. The values were shown as dot plots and medians. a–bBars in the same tissue with the different superscripts indicate significantly different among three groups.
Fig 7.
Pharyngeal shedding of challenge strain and chlamydial loads in lungs after challenge.
The number of chlamydial inclusions in each group (n = 10 for CEF control group and parental HVT group, n = 15 for rHVT-pmpD-N group) was counted in five randomly selected microscopic fields as described in M&M section. The values were shown as dot plots and medians. (A) Median score of pharyngeal shedding of C. psittaci CB7 strain. (B) Chlamydial loads in lungs after challenge. a–bBars in the lungs or pharyngeal swabs with the different superscripts are significantly different.