The development of a chronic model of Haemophilus influenzae infection in the rat

Stephanie Arnold1, Ellena Growcott2, Colin Osborne2. 1Royal Veterinary College, University of London, London, UK, 2Novartis Institutes for BioMedical Research Inc, Emeryville, CA, USA

Haemophilus influenzae (H.i.) is a key causative agent of respiratory tract infection; for example, H.i. is one of the common bacterial agents associated with lung infection in cystic fibrosis (CF), especially in younger patients. As rodent lung infection models can be an important tool for investigating disease progression and new therapies, the objective of this study was to develop a rat model of chronic lung infection with H.i.

H.i was mixed with molten agar noble and spun into heated mineral oil with 0.01% v/v of the surfactant Span® 80 to produce agar beads. Under isofluorane anaesthesia, (day 0), male Wistar(Han) rats (170-220 g; n=6/group) were inoculated (i.t.) with agar beads containing 107 colony forming units (cfu) of H.i. Sham groups were given sterile beads. On days 2, 5, 9 and 14 post infection (p.i.), animals were euthanized by CO2 asphyxiation and the lungs removed for bacterial quantification. Cell counts and ELISAs were performed on bronchoalveolar lavage fluid (BALF) on days 2 & 5 p.i.. To assess antibiotic efficacy animals were dosed on day 1 with 5, 20 or 80 mg/kg ciprofloxacin s.c. BID or sterile water and cfu/lung was determined 24 hours later. Data are expressed as mean ± S.D. and analysed using a Kruskal-Wallis test with a Dunn’s posttest.

Two day p.i the cfu/lung was log10 7.7±0.3 and this was maintained 5 days p.i. (log10 7.2±0.2). At day 9 and 14 p.i. cfu/lung was log10 6.5±0.6 and log10 5.9±0.5 respectively. Total cell counts in BALF were significantly higher than the control group at day 2(Table 1) and this was associated with an elevated neutrophil count. The increased neutrophil count was also observed on day 5.

Table 1: BALF total inflammatory cell number and total neutrophil number for control and H.influenzae infected animals. Results are mean ± SD; *, P<0.01, ** P<0.001, ** P<0.0001 versus control.

In efficacy studies, treatment with ciprofloxacin was effective at reducing bacterial load with even the lowest tested dose of 5 mg/kg resulting in a 2.0±0.2 log reduction compared to vehicle-treated animals.

These results demonstrate that H.i. embedded within agar beads can cause a persistent infection with bacteria measured in rat lungs 14 days p.i. The infection could be treated with ciprofloxacin, a clinically used antibiotic. This model could now be utilized to assess long term infection and the effects of novel therapeutics against H.i. in vivo.