Modeling the catarrhal stage of Bordetella pertussis upper respiratory tract infections in mice

Abstract

Pertussis (whooping cough) is a highly transmissible human respiratory disease caused by Bordetella pertussis, a human-restricted pathogen. Animal models generally involve pneumonic infections induced by depositing large numbers of bacteria in the lungs of mice. These models have informed us about the molecular pathogenesis of pertussis and guided development of vaccines that successfully protect against severe disease. However, they bypass the catarrhal stage of the disease, when bacteria first colonize and initially grow in the upper respiratory tract. This is a critical and highly transmissible stage of the infection that current vaccines do not prevent. Here, we demonstrate a model system in which B. pertussis robustly and persistently infects the nasopharynx of TLR4-deficient mice, inducing localized inflammation, neutrophil recruitment and mucus production as well as persistent shedding and occasional transmission to cage mates. This novel experimental system will allow the study of the contributions of bacterial factors to colonization of and shedding from the nasopharynx, as occurs during the catarrhal stage of pertussis, and interventions that might better control the ongoing circulation of pertussis.

Keywords: Bordetella pertussis; Catarrhal stage; Mouse; Shedding; TLR4 receptor.

Fig. 1.

Distribution of B. pertussis across respiratory organs following inoculation via the conventional high volume ‘pneumonic’ inoculation versus a low volume ‘nasopharyngeal’ inoculation. (A) The graph shows the distribution of B. pertussis in the respiratory tracts of mice 2 h after being inoculated with 50 µl (pneumonic, gray columns) or 5 µl (nasopharyngeal, white columns). n=5 mice per group. Data are mean±s.e.m. ***P<0.001 (two-way ANOVA). Dotted line represents the limit of detection. Experiment was conducted once. (B) The colonization profile of the catarrhal model, with graphs depicting the colonization loads (CFU) in respiratory organs (nasal cavity, trachea and lungs) of mice over time following inoculation with 2.5×10⁷ CFU B. pertussis in 5 µl PBS (‘catarrhal’ model). Filled circles represent the mean±s.d. (n=4). Dotted line represents the limit of detection. Experiment was conducted twice.

Fig. 2.

B. pertussis-induced histopathology of mouse nasal cavity. (A,B) Representative images from control (A) and B. pertussis-challenged (B) mice obtained from Hematoxylin and Eosin (HE)-stained whole-slide scanned images of coronal section of the nose at the level of the eyes and olfactory bulb of the brain (Br). Scale bars: 2 mm (middle panels). Compared to controls (A), mice inoculated with B. pertussis (B) have large numbers of neutrophils, mixed with mucus, proteinaceous material and bacteria filling the nasal meatus (asterisks). In A, the top left image illustrates the dorsal nasal meatus (asterisk) and its close association to the olfactory bulb of the brain (Br). Scale bar: 200 µm. The top right image illustrates a higher magnification of the cribriform plate (arrows) between the brain (Br) and the nasal meatus (asterisk). Scale bar: 100 µm. The bottom left image illustrates the mid- and ventral nasal meatus (asterisk). Scale bar: 200 µm. The bottom right image illustrates a higher magnification of the mid-nasal meatus (asterisk). Scale bar: 100 µm. In B, the top left image illustrates a mucopurulent exudate (arrowheads) within the dorsal nasal meatus (asterisk). Scale bar: 200 µm. The top right image illustrates a higher magnification of the cribriform plate (arrows) between the brain (Br) from the nasal meatus (asterisk). Increased number of inflammatory cellular infiltrates (arrowheads) obscure the nasal mucosa (NM) and extend along the perivascular spaces through the cribriform plate into the olfactory bulb of the brain (Br). Scale bar: 100 µm. The bottom left image illustrates the mid- and ventral nasal meatus (asterisk) filled with mucopurulent exudate (arrowheads). Scale bar: 200 µm. The bottom right image illustrates a higher magnification of the mid-nasal meatus (asterisk) containing mucopurulent exudate. Scale bar: 100 µm. The insert at the top represents a higher magnification of mucopurulent exudate mixed with bacteria (arrowhead). Scale bar: 10 µm. See Fig. S2 for additional details.

Fig. 3.

Upregulation of gene transcripts during the catarrhal stage detected by RT-qPCR. Graph shows comparative expression levels of genes in naïve uninfected mice (PBS-treated; light gray columns) compared with high dose nasopharyngeal inoculation with B. pertussis infection (black columns). Results show strong upregulation of Chil1 and CD177 genes compared to PBS-treated mice 3 days post infection. Moderate but not significant upregulation of C3 and Muc4 genes were observed. In contrast, expression of Muc1 and Muc5ac was unchanged. n=4 mice. ****P<0.0001 (two-way ANOVA). Experiment was conducted twice.

Fig. 4.

Shedding profile of catarrhal stage infection. B. pertussis CFUs recovered on indicated days from the nose tips of C3H/HeJ (black circles, n=10) and C57BL/6J (blue squares, n=5) mice inoculated with 5×10⁷ CFU of B. pertussis delivered in 5 μl PBS. Data are mean±s.d. Experiment was conducted twice.

Fig. 5.

Transmission of B. pertussis among adult mice and seropositivity of anti-B. pertussis IgG in exposed mice. (A) Filled circles on graph represent the number of B. pertussis CFUs recovered from the nasal cavities of individual mice that had been inoculated with B. pertussis (Inoculated) or co-housed with the inoculated mice (Exposed) for 28 days (two inoculated+two naïve; n=5 cages). Horizontal bar indicates the mean. Dashed line indicates the level of detection. Experiment was conducted twice. (B) The graph shows the relative titers of anti-B. pertussis IgG antibodies detected in individual sera of 10 exposed mice co-housed in five cages (from A) for 28 days with mice inoculated via catarrhal model (see text for description). Asterisk indicates culture-positive mice identified in A. Experiment was conducted twice.