Mucosal Vaccination with Live Attenuated Bordetella bronchiseptica Protects against Challenge in Wistar Rats

Abstract

Bordetella bronchiseptica (Bb) is a Gram-negative bacterium responsible for canine infectious respiratory disease complex (CIRDC). Several vaccines targeting this pathogen are currently licensed for use in dogs, but their mechanism of action and the correlates of protection are not fully understood. To investigate this, we used a rat model to examine the immune responses induced and the protection conferred by a canine mucosal vaccine after challenge. Wistar rats were vaccinated orally or intranasally on D0 and D21 with a live attenuated Bb vaccine strain. At D35, the rats of all groups were inoculated with 10³ CFU of a pathogenic strain of B. bronchiseptica. Animals vaccinated via either the intranasal or the oral route had Bb-specific IgG and IgM in their serum and Bb-specific IgA in nasal lavages. Bacterial load in the trachea, lung, and nasal lavages was lower in vaccinated animals than in non-vaccinated control animals. Interestingly, coughing improved in the group vaccinated intranasally, but not in the orally vaccinated or control group. These results suggest that mucosal vaccination can induce mucosal immune responses and provide protection against a Bb challenge. This study also highlights the advantages of a rat model as a tool for studying candidate vaccines and routes of administration for dogs.

Keywords: Bordetella bronchiseptica; canine infectious respiratory disease complex (CIRDC); live attenuated vaccines; mucosal vaccination; rat model.

Figure 1

Study design and humoral responses after vaccination and challenge. Study design of vaccination and challenge model of Wistar rats. Animals were vaccinated intranasally or orally at D0 and D21 and challenged at D35 (A). Intranasal and oral vaccination induced systemic and mucosal anti-B. bronchiseptica vaccine IgG, IgM, and IgA. Blood was collected 13 days post-boost, and 7- and 14-days post challenge upon euthanasia via the intracardiac route. Feces and nasal lavages were collected at the same timepoint. Anti-Bb vaccine IgG and IgM were measured in serum and differences in the OD were compared among groups and timepoints (B,C). Anti-Bb vaccine IgA was measured in nasal lavages and OD was compared between groups and timepoints (D). The results are shown with the OD and mean with SDs are presented. (n = 4 per group per day). p values were determined using two-way ANOVA (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 2

Intranasal and oral administration reduces bacterial burden in lung, trachea and nasal lavages after challenge and vaccine strain persist in the respiratory tract up to 28 days after boost. Briefly, 13 days after the boost and 7 and 14 days after challenge, the presence of challenge and vaccine strain in the lung, trachea and nasal lavages of euthanatized rats was determined via qPCR. Challenge strain was only detected in the unvaccinated rats 7 and 14 days after challenge in the lung, trachea, and nasal lavages (A). The vaccine strain was detected in the vaccinated animals and compared between different days in each organ (B). Ct values were obtained via real-time qPCR analysis. Each dot represents an individual rat. The error bars represent the standard error of the mean (n = 4 per group). The asterisks and brackets refer to statistical significance determined via ANOVA with multiple comparisons. (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Figure 3

Mucosal and systemic antibodies correlate with lower bacterial burden in the respiratory tract. Correlograms values were calculated with R. Unvaccinated rats 7 and 14 days after challenge showing a strong negative correlation between the colonization and the antibodies (A). Antibodies of vaccinated animals which were positive but not strongly correlated with the vaccine presence in the respiratory tract (B). Positive correlations appear as blue circles and the negative correlations appear from orange to red. The size of the circle indicates the strength of the correlation.

Figure 4

Intranasal vaccination of live-attenuated B. bronchiseptica vaccine decreases cough in infected rats. Coughs were counted and recorded in three animals per groups and three times after challenge (6-, 9- and 12 days p.c.), for 10 min each. Represents the number of coughs per day per each group and the mean per group and day were compared between each other (A). The number of coughs per animal are presented day by day (B). The accumulated cough of the three days was summed up and compared between groups (C). p values were determined by two-way ANOVA (* p ≤ 0.05).

Figure 5

Signs of inflammation in the lung and trachea after challenge. Lung and trachea of each animal after challenge were stained with hematoxylin and eosin (H&E) and images are displayed at 20× magnification. Lesions of the trachea of vaccinated and unvaccinated animals after challenge are shown (A). Lesions of the lung of vaccinated and unvaccinated animals after challenge are shown (B). Four animals per group.

Figure 6

Oral vaccination leads to increase in Th17:Treg ratio of cell within splenic T-cells 7 days after challenge. Percentage of parent of CD3+CD4+, CD3+CD8+ and double-negative and doble-positive T-cells (A). Ratio of the percentage of CD4+ cells to the percentage of CD8+ cells within the CD3+ population (B). Percentage of parent and dot plots of CD3+CD4+IL17+ (Th17) and CD3+CD4+FOXP3+CD25+ (Treg cells) (C). Ratio of the percentage of parent of CD3+CD4+IL17+ to the percentage of parent of CD3+CD4+FOXP3+CD25+ (D). Each dot represents an individual rat. The error bars represent the standard error of the mean (n = 4 per group). The asterisks and brackets refer to statistical significance determined via ANOVA with multiple comparisons; * p ≤ 0.05.