Fig 1.
B. pseudohinzii colonizes the respiratory tract and middle ears of C57BL/6J mice.
1. Mice inoculated with 7,500 CFU of B. pseudohinzii (n = 4 per group) were sacrificed at days 3, 7, 21, 49, and 70 post inoculation. B. pseudohinzii numbers recovered from the nasal cavity, trachea, lungs and left and right middle ear bullae are presented for each day. Bacteria in the middle ears were bilaterally distributed in equal numbers (lower panel, red squares- left ear; blue squares- right ear). Error bars indicate standard deviation. Dotted lines indicate limit of detection.
Fig 2.
Histopathology of mouse nasal cavity and middle ears on Bordetella pseudohinzii colonization.
Histopathology analyses conducted on C57BL/6J mice inoculated with 7,500 CFU of B. pseudohinzii delivered in 25 μl of PBS. Groups of infected mice (n = 6) were sacrificed on 3, 14, 28 and 123- days post inoculation (DPI). (A-D) Coronal sections of the nasal cavity of C57BL/6J mice stained with Hematoxylin and Eosin (HE) stain. 3 DPI. Image of nasal meatus showing small amounts of exudate at the frontonasal sinus (arrow). Scale bar = 500 μm. B) Higher magnification of Fig A (dashed rectangle) showing neutrophils embedded in pale basophilic mucopurulent exudate (arrow). Scale bar = 100 μm. C) 7 DPI. Image of nasal meatus with mild amounts of exudate (asterisk) and a thin basophilic, film-like layer extending throughout the nasal meatus (arrows and inset). Scale bar = 500 μm; Inset Scale bar = 10 μm. D) Higher magnification of Fig C, (dashed rectangle), showing mild amounts of neutrophils embedded in pale basophilic material (arrow). Scale bar = 100 μm. Abbreviations: Nasal meatus (NM); Olfactory bulb (OB); Nasopharynx (NP). (E-J) HE stained transverse sections through the middle ear of C57BL/6J mice controls (E, F) and infected with Bordetella pseudohinzii (G-J). E) 3 DPI showing Eustachian tube (long arrow), round window membrane (short arrow), and tympanic membrane (arrowheads). Scale bar = 500 μm. F) Higher magnification of E, (dashed rectangle area), showing the middle ear tympanic bulla and thin layer of ciliated epithelia (mucoperiosteum), overlying a thin lamina propria (arrowheads). Scale bar = 50 μm. G) 14 DPI showing large accumulation of exudate extending from Eustachian tube (long arrow), tympanic membrane (arrowheads), middle ear bones (asterisk), and round window (short arrow). Scale bar = 500 μm. H) Higher magnification of G, (dashed rectangle), Middle ear and tympanic bulla showing diffusely hypercellular mucoperiosteum (arrowheads) and hypertrophic mesenchymal cells (arrow) covering the lamellar bone (LM). Scale bar = 50 μm. Inset: Gram-negative (red) bacteria (asterisk) attached to cilia of the mucoperiosteum. Gram stain. Scale bar = 10 μm. I) 28 DPI showing moderate amounts of exudate in the middle ear and adjacent structures. Arrowhead points to the tympanic membrane, and arrow points to the mucoperiosteum. Scale bar = 500 μm. J) Higher magnification of Fig. I, (dashed rectangle), showing middle ear and tympanic bulla showing hypercellular mucoperiosteum (arrowhead) and lymphoid tissue formation (arrow). Scale bar = 50 μm. Abbreviations: External auditory meatus (EAM); Middle Ear (ME); Cochlea (Co); Brain Stem (BS); Lamellar bone (LB); Bone marrow (BM). (K-P) HE stained transverse sections through the middle ear of C57BL/6J mice 123 DPI with Bordetella pseudohinzii. K) Image shows focal granulomatous inflammation about the round window (arrowhead) and malleolus (asterisk). Tympanic membrane (arrow). Scale bar = 500 μm. L) Higher magnification of K, (dashed rectangle area), showing the round window with an area of granulomatous inflammation that contains cholesterol clefts (arrow) mixed with macrophages and neutrophils. Scale bar = 50 μm.
Fig 3.
B. pseudohinzii colonization causes measurable hearing loss in C57BL/6J mice.
Auditory brainstem response (ABR) thresholds (panel A and B) and distortion product otoacoustic emission (DPOAE) levels (panel C and D) recorded every 3–7 days in mice inoculated with 30 CFU (low dose—panel A and C) or 7500 CFU (high dose- panel B and D) of B. pseudohinzii. Selected time points from individual animals are shown for clarity.
Fig 4.
Determining the infectious dose of B. pseudohinzii.
The colonization levels of the nasal cavity (filled circles) and middle ears (clear circles) in groups (n = 4) of mice 7 days after being inoculated from the external nares with serial dilutions of an estimated 8, 4, 2 and 1 CFU of B. pseudohinzii in 5 μl PBS.
Fig 5.
Colonization of mice by B. pseudohinzii following experimental inoculation or natural transmission.
A.) Colonization profiles of B. pseudohinzii in the nasal cavity, trachea, lungs and middle ears of C57BL/6J mice experimentally inoculated with B. pseudohinzii (5 CFU in 5 μl) and analyzed on days 1, 3, 7, 21, 49, and 98 days post inoculation (n = 3 per group). Error bars indicate standard deviation. Dashed line indicates the limit of detection. B.) Mice colonized via natural transmission when co-housed for 28 days with experimentally inoculated mice. Colonization profiles for the individual mice are shown in the lower graphs (NC: nasal cavity, TR: trachea, LNG: lungs, RE/LE: right and left middle ear bullae).
Fig 6.
The study of B. pseudohinzii pathogenesis in the middle ear.
Figure schematically depicts the timeline in the pathogenesis of B. pseudohinzii induced otitis media in C57BL/6J mice. The bacterial numbers are derived from bacteria recovered from the middle ears, and pathology severity scores [44] are based on histological observations of experimentally infected animals [Histopathologic severity grades—0 (no significant histopathological alterations); 1 (minimal); 2 (mild); 3 (moderate); or 4 (severe)]. Letters (a-g) indicate approximate points where the critical stages in the development of chronic otitis media begin, and which are amenable to study using the B. pseudohinzii-mouse infection model. [a: Nasopharyngeal colonization, b: Ascent of the Eustachian tube, c: Colonization of middle ear, d: Induction of acute inflammatory stage, e: Inflammation and mucoidal effusion, f: Acute inflammatory stage, g: Chronic infection stage].
Fig 7.
Adaptive immune components contribute to controlling B. pseudohinzii colonization in the middle ears of mice.
Numbers of B. pseudohinzii recovered from wild type (C57BL/6J) and B-cell (MuMt-), T-cell (Tcratm1Mom), and B/T-cell (Rag-/-) deficient mice 49 days following inoculation with 7500 CFU delivered to the external nares in 25 μl of PBS. Error bars indicate standard deviation. Arrows indicate the contributions the adaptive immune components have on controlling colonization levels. One-way Anova with Dunnet’s post-test was used to determine statistical difference compared to wild-type: † = p-value <0.1, * = p-value <0.05, ** = p-value<0.002.