Challenge protocol time table.

<p>Protocols to assess the ability of <i>M. catarrhalis</i> to induce OM in RSV-compromised juvenile chinchillas. For both protocols, nasopharyngeal mucosae, Eustachian tubes and middle ear mucosae were collected for culture at the time of sacrifice. Bullar washes were performed upon bisection of the bullae to recover planktonic <i>M. catarrhalis</i>.</p

Detection of <i>M. catarrhalis</i> in the chinchilla middle ear.

<p>Immunohistochemistry of a chinchilla ET 11 days after challenge with <i>M. catarrhalis</i>. <i>M. catarrhalis</i> (red) was found associated with ciliated cells in both A) the middle ear and B) the ET. Images collected at100X. Rabbit anti-<i>M. catarrhalis</i> WCL = whole cell lysate.</p

Detection of <i>M. catarrhalis</i> and NTHI in biofilms recovered from the chinchilla middle ear.

<p>Immunofluorescence image of a biofilm incubated with rabbit-anti NTHI outer membrane protein P5 (green), mouse anti-<i>M. catarrhalis</i> PilA (red), and stained with DAPI to label DNA (blue). A) Merged image, B) <i>M. catarrhalis</i>, C) NTHI, D) serial section control demonstrating no labeling of either <i>M. catarrhalis</i> or NTHI. Scale bar = 5 µm.</p

Detection of <i>M. catarrhalis</i> in the chinchilla middle ear.

<p>Proof of concept study: Scanning electron microscopy (SEM) images of chinchilla middle ear mucosa 11 days after intranasal <i>M. catarrhalis</i> challenge. <i>M. catarrhalis</i> (pseudo-colored purple) was detected on the mucosal surface in association with (A) cilia and also (B) with an elevated area of membrane that was ruffled in appearance.</p

Clinically-relevant assessment for the development of experimental OM as determined by video otoscopy and tympanometry.

<p>Expanded study: Intranasal challenge of chinchillas with NTHI, <i>M. catarrhalis</i> and RSV resulted in clinical indicators of otitis media as determined by otoscopy and tympanometry. (A) A healthy chinchilla tympanic membrane (TM), with both the annulum (a) and the umbo of the malleus (U) labeled. (B) Seven days after <i>M. catarrhalis</i> challenge, erythema (arrow) was observed along the umbo, along with darkening of the TM. Panels C-E – TM 11, 14 and 17 days after <i>M. catarrhalis</i> challenge, respectively, erythema (arrows) was observed along the annulum, over the U and across the TM (*).</p

<i>M. catarrhalis</i>-induced inflammation within the chinchilla middle ear.

<p>Proof of concept study: Intranasal challenge of chinchillas with <i>M. catarrhalis</i> and RSV resulted in inflammation within the middle ear. (A) Seven days after bacterial challenge vasodilatation (†) was evident, as was focal submucosal edema (arrow) and erythema (*). Hemorrhagic foci (Δ) were present in the inferior bulla as well as near the opening of the Eustachian tube. (B) Eleven days after bacterial challenge bullous myringitis (box and inset) developed in addition to vasodilatation (†), erythema (*), hemorrhagic foci (Δ) and blood in the opening of the ET (‡). TM = tympanic membrane, S = septa, C = cochlea.</p

<i>M. catarrhalis</i> was detected adherent to multiple mucosal tissues.

<p>Expanded study: Percentage of mucosal sites that were culture-positive for <i>M. catarrhalis. M. catarrhalis</i> was detected in the middle ear as early as seven days and up to 17 days after intranasal challenge with <i>M. catarrhalis</i>.</p

Gross morphological changes within the chinchilla middle ear after co-challenge with NTHI and <i>M. catarrhalis</i>.

<p>Expanded study: Chinchillas challenged intranasally with NTHI, <i>M. catarrhalis</i> and RSV demonstrated changes in the middle ear mucosa. Panels A-D: Seven, eleven, fourteen and seventeen days after challenge with <i>M. catarrhalis</i>, respectively, vasodilatation (†) and erythema (*) were evident. Bullous myringitis (encircled) was evident at days 7, 11 and 17 (panels A, B and D), and hemorrhagic foci (Δ) were present at day 14 and 17 (panels C and D). Moreover, 4 out of 16 animals (25%) exhibited blood (‡) in the opening of the Eustachian tube on days 11 and 14 (panels B and C). TM = tympanic membrane, S = septa, C = cochlea, ET = Eustachian tube.</p