Blood for serum was collected via cardiac puncture prior to immunization (pre) and one week after receipt of the second immunizing dose (immune) for all animals immunized via a preventative regimen. Animals immunized via a therapeutic regimen were bled prior to NTHI challenge and at study end.
NP lavages were performed on animals immunized via a preventative regimen prior to immunization, one week after receipt of the final dose and on days 3, 7, 10, and 14 after bacterial challenge by passive inhalation of 500 μl sterile pyrogen-free saline as previously described 24 (link).
Video otoscopy (using a 0°, 3-in. probe connected to a digital camera system) to monitor signs of tympanic membrane inflammation and/or presence of fluid within the middle ear space was performed and overall signs of OM were rated on a scale of 0 to 4+ as previously described 7 (link), 24 (link), 31 (link) . Middle ears with a score of ≥ 2.0 were always considered positive for OM as MEF is visible behind the tympanic membrane. Each middle ear was considered independent, and for each cohort, the percentage of middle ears with OM was calculated.
Epitympanic taps to retrieve middle ear effusions were performed on any chinchilla whose tympanic membrane was rated as ≥ 2.5 on a scale of 0 to +4.0. Epitympanic taps were not performed on ears ranked 2.0, due potential for perforation of the tympanic membrane to retrieve the low volume of MEF. NP lavage and middle ear fluids were serially diluted and plated onto chocolate agar supplemented with 15 μg ampicillin/ ml medium or chocolate agar, respectively, to semi-quantitate CFU NTHI per ml fluid type. The mean CFU NTHI/ ml fluid was reported for each cohort.
NP lavages were performed on animals immunized via a preventative regimen prior to immunization, one week after receipt of the final dose and on days 3, 7, 10, and 14 after bacterial challenge by passive inhalation of 500 μl sterile pyrogen-free saline as previously described 24 (link).
Video otoscopy (using a 0°, 3-in. probe connected to a digital camera system) to monitor signs of tympanic membrane inflammation and/or presence of fluid within the middle ear space was performed and overall signs of OM were rated on a scale of 0 to 4+ as previously described 7 (link), 24 (link), 31 (link) . Middle ears with a score of ≥ 2.0 were always considered positive for OM as MEF is visible behind the tympanic membrane. Each middle ear was considered independent, and for each cohort, the percentage of middle ears with OM was calculated.
Epitympanic taps to retrieve middle ear effusions were performed on any chinchilla whose tympanic membrane was rated as ≥ 2.5 on a scale of 0 to +4.0. Epitympanic taps were not performed on ears ranked 2.0, due potential for perforation of the tympanic membrane to retrieve the low volume of MEF. NP lavage and middle ear fluids were serially diluted and plated onto chocolate agar supplemented with 15 μg ampicillin/ ml medium or chocolate agar, respectively, to semi-quantitate CFU NTHI per ml fluid type. The mean CFU NTHI/ ml fluid was reported for each cohort.
