Nasopharyngeal colonization with nontypeable Haemophilus influenzae in chinchillas

Abstract

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.

FIG. 1

SDS-PAGE and immunoblot analysis of bacteria recovered from chinchilla nasal lavage fluids or middle ear effusion. (A) SDS-PAGE was performed on a 12.5% polyacrylamide gel, and proteins were visualized by Rapid Coomassie blue staining. Lanes: 1, prestained molecular weight markers; 2, parent NTHI 12; 3, streptomycin-resistant strain 12 generated through spontaneous mutation. (B and C) Young chinchillas (2 to 3 months old) were challenged intranasally with freshly cultured streptomycin-resistant NTHI 12 as described in Materials and Methods. NP lavage and aspiration of middle ear fluid were performed 4 days after bacterial inoculation. Lanes: 1, prestained molecular weight markers; 2, whole-cell lysate of streptomycin-resistant NTHI 12 inoculum; 3 and 4, whole-cell lysate of bacteria recovered from nasal lavage fluids in the presence of streptomycin; 5 and 6, whole-cell lysate of bacteria recovered from middle ear fluids in the absence of streptomycin; 7 and 8, whole-cell lysate of bacteria recovered from the same middle ear fluids in the presence of streptomycin. Proteins were visualized by Rapid Coomassie blue staining (B), and immunoblotting was performed with guinea pig anti-P6 antiserum (C).

FIG. 2

SDS-PAGE and immunoblot analysis of anti-chinchilla IgA and IgG antibodies. SDS-PAGE was performed on a 12.5% polyacrylamide gel under reducing conditions (4% 2-mercaptoethanol). Lanes: 1, prestained molecular weight markers; 2, purified chinchilla IgA; 3, purified chinchilla IgG. Proteins were visualized by Rapid Coomassie blue staining (A), and immunoblotting of samples in panel A was performed with guinea pig anti-α chain of chinchilla IgA (B) or guinea pig anti-chinchilla IgG (C).

FIG. 3

Tympanograms of mature and young chinchillas. The animals were challenged intranasally with freshly cultured streptomycin-resistant NTHI 12 as described in Materials and Methods. The development of middle ear infection was monitored by otoscopic examination and tympanometry. Tympanograms were recorded before bacterial challenge (A and C) and again at 4 days postinoculation (B and D).

FIG. 4

SDS-PAGE and immunoblot analysis of Ig responses in chinchilla convalescent-phase and immune sera. SDS-PAGE was performed on a 12.5% polyacrylamide gel. Lanes: 1, prestained molecular weight markers; 2, whole-cell lysate of NTHI 12; 3, HMW proteins (HMW1 + HMW2) purified from NTHI 12; 4, P6 protein purified from Hib Eagan. Proteins were visualized by Rapid Coomassie blue staining (A), and immunoblotting was performed with chinchilla convalescent-phase sera (B) or immune sera collected from animals immunized i.m. with inactivated NTHI 12 whole cells (C) or with NTHI LCDC2 (D) or normal chinchilla sera (E).