THE JOUR;\,AL OF INFECTIOUS DISEASES • VOL. 136, SUPPLEMENT. AUGUST 1977 © 1977 by the University of Chicago. All rights reserved.
Comparison of Otitis Media Due to Types 3 and 23 Streptococcus pneumoniae in the Chinchilla Model From the Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, Minnesota
G. Scott Giebink and Paul G. Quie
Acute and recurrent otitis media remain a major health problem among children and cause significant morbidity despite the use of antibiotics [1, 2]. Streptococcus pneumoniae is the most frequent bacterial pathogen implicated in this disease and may be responsible for recurrent otitis media [1]. Two-thirds of the pneumococcal isolates from the middle ears of children with acute otitis media belong to six serotypes. In order of decreasing frequency these are serotypes 19, 23, 6, 14, 3, and 18 [3]. We have reported previously a chinchilla model for pneumococcal otitis media that parallels the immunopathogenesis of pneumococcal otitis media in children [4, 5]. The present report compares the similarities and differences of type 3 and type 23 pneumococcal otitis media in the chinchilla model.
Materials and Methods
Healthy one- to two-year-old chinchillas were infected with encapsulated S. pneumoniae type 3 or 23 by inoculation of a bacterial suspension These studies were supported by contract no. NOI-AI52533 from the National Institute of Allergy and Infectious Diseases. Please address requests for reprints to Dr. G. Scott Giebink, Department of Pediatrics, University of Minnesota Hospitals, Box 483 Mayo, Minneapolis, Minnesota 55455.
through the tympanic membrane into the middle ear fossa as previously described [5]. Virulence of the organisms was maintained by monthly passage in mice. Pneumococci were grown in Todd-Hewitt broth (Difco, Detroit, Mich.) enriched with 3% bovine serum albumin; I % glucose was added during the final 2 hr of incubation. Bacteria were washed in phosphate-buffered saline (PBS, pH 7.4), and the suspension was adjusted to a known bacterial concentration by spectrophotometry. The pneumococcal suspension was diluted in PBS to a concentration of 102-108 bacteria / ml for inoculation into chinchillas. Middle ear infection was initiated by inoculation of 0.3 ml of the diluted bacterial suspension through each tympanic membrane with a 2inch, 25-gauge needle on a tuberculin syringe with use of the operating microscope. For this procedure chinchillas were anesthetized with ether and were awake within 5 min. The chinchillas were sacrificed for examination of the middle ear fossa, and cultures and blood samples were obtained. Animals were anesthetized with 0.5 ml of 25% urethane/ l Otl g of body weight given ip. Cerebrospinal fluid was obtained by exposure of the allantooccipital membrane and withdrawal of fluid with a tuberculin syringe; 0.1 ml of cerebrospinal fluid was plated directly onto sheep blood agar for culture. Blood was obtained asceptically by cardiac puncture for culture, and serum was frozen at -70 C for serologic
S191
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 13, 2015
Only a few pneumococcal serotypes are responsible for the majority of cases of acute otitis media due to Streptococcus pneumoniae in children. The immunopathogenesis of otitis media caused by two serotypes, types 3 and 23, was studied in chinchillas. Chinchillas with type 23 pneumococcal otitis media were capable of clearing the infection over a six-week period without treatment, whereas the type 3 infection persisted throughout the six-week study. In addition, bacteremia or meningitis occurred more frequently and earlier with type 3 pneumococcal otitis media than with type 23. The resolution of the type 23 pneumococcal infection paralleled the development of type-specific antibody, as measured by radioimmunoassay, whereas type 3 infection was associated with a fall in serum antibody to low levels. Since the pathogenesis of pneumococcal otitis media in the chinchilla differs between pneumococcal serotypes, it may be important to correlate pneumococcal serotypes with sequelae and recurrences of otitis media in children.
Sl92
Giebink and Quie
Table 1. Results of middle ear cultures from chinchillas with experimentally induced otitis media due to types 3 and 23 Streptococcus pneumoniae, Infection, interval between inoculation and sacrifice. (days)
No. of animals with positive culture [oo, sacrificed (%)*
S. pneumoniae type 23 1-3 4-7 9-14 17-21 42 S. pneumoniae type 3
7/8 (98) 47/47 (100) 29/34 (85) 9/14 (64) 1/7 (14) 8/9 (89) 22/29 (76) 5/8 (63) 4/4 (100) 6/6 (100)
14 21 42
"Number of animals with positive culture represents the number of animals with middle ear cultures positive for S. pneumoniae type 3 or 23 at the time of sacrifice.
studies. All cultures were plated directly onto 5% sheep blood agar. Blood was cultured aerobically in Columbia broth (Baltimore Biological Laboratories, Cockeysville, Md.) and subcultured on sheep blood agar. S. pneumoniae were identified morphologically and by the type-specific Quellung reaction. Assays of pneumococcal antibody in serum
Results
Type 23 pneumococcal otitis media. The middle ears of 115 chinchillas were inoculated with type 23 S. pneumoniae. Five of the inoculated animals died. Bilateral middle ear cultures were obtained from the remaining 110 animals at intervals between days I and 42 after inoculation (table 1). Pneumococcal otitis media was evident in 98% of the animals sacrificed during the first week. Of the animals sacrificed between days 9 and 14, 15
Comparison of Otitis Media Due to Types 3 and 23 Streptococcus pneumoniae in the Chinchilla Model From the Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis, Minnesota
G. Scott Giebink and Paul G. Quie
Acute and recurrent otitis media remain a major health problem among children and cause significant morbidity despite the use of antibiotics [1, 2]. Streptococcus pneumoniae is the most frequent bacterial pathogen implicated in this disease and may be responsible for recurrent otitis media [1]. Two-thirds of the pneumococcal isolates from the middle ears of children with acute otitis media belong to six serotypes. In order of decreasing frequency these are serotypes 19, 23, 6, 14, 3, and 18 [3]. We have reported previously a chinchilla model for pneumococcal otitis media that parallels the immunopathogenesis of pneumococcal otitis media in children [4, 5]. The present report compares the similarities and differences of type 3 and type 23 pneumococcal otitis media in the chinchilla model.
Materials and Methods
Healthy one- to two-year-old chinchillas were infected with encapsulated S. pneumoniae type 3 or 23 by inoculation of a bacterial suspension These studies were supported by contract no. NOI-AI52533 from the National Institute of Allergy and Infectious Diseases. Please address requests for reprints to Dr. G. Scott Giebink, Department of Pediatrics, University of Minnesota Hospitals, Box 483 Mayo, Minneapolis, Minnesota 55455.
through the tympanic membrane into the middle ear fossa as previously described [5]. Virulence of the organisms was maintained by monthly passage in mice. Pneumococci were grown in Todd-Hewitt broth (Difco, Detroit, Mich.) enriched with 3% bovine serum albumin; I % glucose was added during the final 2 hr of incubation. Bacteria were washed in phosphate-buffered saline (PBS, pH 7.4), and the suspension was adjusted to a known bacterial concentration by spectrophotometry. The pneumococcal suspension was diluted in PBS to a concentration of 102-108 bacteria / ml for inoculation into chinchillas. Middle ear infection was initiated by inoculation of 0.3 ml of the diluted bacterial suspension through each tympanic membrane with a 2inch, 25-gauge needle on a tuberculin syringe with use of the operating microscope. For this procedure chinchillas were anesthetized with ether and were awake within 5 min. The chinchillas were sacrificed for examination of the middle ear fossa, and cultures and blood samples were obtained. Animals were anesthetized with 0.5 ml of 25% urethane/ l Otl g of body weight given ip. Cerebrospinal fluid was obtained by exposure of the allantooccipital membrane and withdrawal of fluid with a tuberculin syringe; 0.1 ml of cerebrospinal fluid was plated directly onto sheep blood agar for culture. Blood was obtained asceptically by cardiac puncture for culture, and serum was frozen at -70 C for serologic
S191
Downloaded from http://jid.oxfordjournals.org/ at University of Lethbridge on September 13, 2015
Only a few pneumococcal serotypes are responsible for the majority of cases of acute otitis media due to Streptococcus pneumoniae in children. The immunopathogenesis of otitis media caused by two serotypes, types 3 and 23, was studied in chinchillas. Chinchillas with type 23 pneumococcal otitis media were capable of clearing the infection over a six-week period without treatment, whereas the type 3 infection persisted throughout the six-week study. In addition, bacteremia or meningitis occurred more frequently and earlier with type 3 pneumococcal otitis media than with type 23. The resolution of the type 23 pneumococcal infection paralleled the development of type-specific antibody, as measured by radioimmunoassay, whereas type 3 infection was associated with a fall in serum antibody to low levels. Since the pathogenesis of pneumococcal otitis media in the chinchilla differs between pneumococcal serotypes, it may be important to correlate pneumococcal serotypes with sequelae and recurrences of otitis media in children.
Sl92
Giebink and Quie
Table 1. Results of middle ear cultures from chinchillas with experimentally induced otitis media due to types 3 and 23 Streptococcus pneumoniae, Infection, interval between inoculation and sacrifice. (days)
No. of animals with positive culture [oo, sacrificed (%)*
S. pneumoniae type 23 1-3 4-7 9-14 17-21 42 S. pneumoniae type 3
7/8 (98) 47/47 (100) 29/34 (85) 9/14 (64) 1/7 (14) 8/9 (89) 22/29 (76) 5/8 (63) 4/4 (100) 6/6 (100)
14 21 42
"Number of animals with positive culture represents the number of animals with middle ear cultures positive for S. pneumoniae type 3 or 23 at the time of sacrifice.
studies. All cultures were plated directly onto 5% sheep blood agar. Blood was cultured aerobically in Columbia broth (Baltimore Biological Laboratories, Cockeysville, Md.) and subcultured on sheep blood agar. S. pneumoniae were identified morphologically and by the type-specific Quellung reaction. Assays of pneumococcal antibody in serum
Results
Type 23 pneumococcal otitis media. The middle ears of 115 chinchillas were inoculated with type 23 S. pneumoniae. Five of the inoculated animals died. Bilateral middle ear cultures were obtained from the remaining 110 animals at intervals between days I and 42 after inoculation (table 1). Pneumococcal otitis media was evident in 98% of the animals sacrificed during the first week. Of the animals sacrificed between days 9 and 14, 15
