635
Neonatal Mouse Model of Group B Streptococcal Infection Ariane K. Rodewald, Andrew B. Onderdonk, Henry B. Warren, and Dennis L. Kasper
Channing Laboratory, Brigham and Women's Hospital, and Division of Infectious Diseases, Beth Israel Hospital. Departments ofMedicine and Pathology. Harvard Medical School. Boston, Massachusetts
Neonatal mice were infected with type III group B streptococcal (GBS) strain M781 by the intraperitoneal route. Age-related susceptibility to challenge was seen within the first 5 days of life. Quantitative blood cultures demonstrated a rapid increase in bacterial numbers during the first 30 h after challenge. Infected pups showed clinical signs of septicemia, and most succumbed within 48 h of challenge. Histopathologic evaluation of the neonates showed bacterial infection within 1 day after challenge. Pregnant adult mice were given a single inoculation of serum raised in rabbits against a tetanus toxoid-conjugated type III GBS polysaccharide vaccine. This serum passively protected 100% of the offspring. This neonatal mouse model of GBS infection and protection may be suitable for study of various forms of intervention.
Received 9 January 1992; revised 6 April 1992. Grant support: National Institutes of Health (AI-23339, AI-30628). Reprints or correspondence: Dr. Ariane Rodewald. Channing Laboratory. 180 Longwood Ave., Boston, MA 02115-5899. The Journal of Infectious Diseases
1992;166:635-9
© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0030$01.00
pathologic and microbiologic features of the experimental disease, as well as the passive protection of neonatal mice after G BS challenge.
Materials and Methods Bacterial strain. GBS type III, strain M781, was originally isolated from a human neonate with GBS meningitis and donated to the Channing Laboratory by C. Baker (Baylor College of Medicine, Houston). Preparation of inoculum. Two or three colonies of strain M781 from a 12-h tryptic soy agar plate (Scott Laboratories, Fiskeville, RI) were inoculated into 10 mL of Todd-Hewitt broth (Difco, Detroit) and grown at 37°C overnight. This starter inoculum was then placed into 250 mL of Todd-Hewitt broth and incubated in a shaker waterbath at 37°C with monitoring of the optical density (OD) at 450 nm until the culture had reached mid-log phase. Bacterial suspensions were diluted to the appropriate viable cell density. on the basis of the OD at 450 nm, as calibrated by previous studies in Todd-Hewitt broth [6]. Mice. Timed-pregnant, CD-I virus antibody-free female mice (Charles River Laboratories, Wilmington, MA) were used for all experiments at 15 or 16 days of gestation. Animals were housed I per cage and received Lab Chow (Purina, St. Louis) and water ad libitum. Mice were watched closely to determine the date of parturition. Blood cultures. Blood was drawn from neonatal pups by percutaneous cardiac puncture with a 27-gauge needle. A 0.02-mL volume of blood was placed into 10 mL of melted (46°C) tryptic soy agar, mixed, and poured into sterile plates. Representative colonies were confirmed as GBS by Gram's stain and CAMP test. Histopathologic examination. Moribund animals were euthanized with diethyl ether and were placed into neutral-buffered 10% formalin for fixation. Paraffin sections were stained with hematoxylin-eosin or with Brown and Hopps stain. Administration ofantiserum to pregnant mice and challenge of neonates. Antiserum was raised in rabbits to the type III GBS
polysaccharide coupled to tetanus toxoid in our laboratory according to previously described methods [7]. This antiserum with an ELISA titer of I:256,000 was administered to pregnant
Downloaded from http://jid.oxfordjournals.org/ at Russian Archive on December 20, 2013
More than 20 years ago, group B streptococci (GBS) were first identified as an important cause of neonatal sepsis and meningitis in humans. The incidence ofGBS infection is 2-3 cases/I 000 live births, with a mortality of r - 20% for infected neonates. Between 25% and 50% of infants surviving neonatal infection continue to show serious neurologic sequelae. Despite improved methods for the early detection of GBS infection and the availability of appropriate antimicrobial agents for treating this disease, a principal issue remains one of prevention. Two-thirds of the cases of GBS disease in the United States are due to capsular type III strains. A correlation has been shown between low levels of maternal antibody to the type III polysaccharide and GBS infection in neonates [l]. Test systems in a number of animals, including adult mice [2, 3], newborn rats [4], and chick embryos [5], have been used in studies of the host immune response to GBS. The use of adult mice for these studies has been problematic, especially when disease is induced with type III GBS, because of lack of virulence in older animals. These in vivo test systems have proven to be either expensive and cumbersome or inadequate in providing end points consistent with the disease as it occurs in human neonates. Although the neonatal rat model more closely simulates human disease, difficulty has been encountered in studying the details of immunity in any species other than the mouse because of the lack ofimmunologic reagents. We have therefore established an animal model system in newborn mice as a practical and inexpensive tool for evaluating both the pathogenesis of the disease and the host immune response. Here we describe the histo-
636
Concise Communications
Results Establishment ofa lethality-dose relationship. To develop a model in mice that simulates GBS septicemia and meningitis in humans, a dose-response experiment was done and the lethal dose of GBS for l-day-old mice (day of birth) was established. Mice of this age were selected to simulate the stage of development at which human neonates with the early-onset form of the disease most frequently become infected. Twenty-four animals were inoculated intranasally with GBS strain M781 at doses of 105-108 cfu in 0.05 mL with no signs of illness. Eighty-three newborn mice received 0.1 mL of broth containing an inoculum ranging from 102 to 108 cfu by the ip route. Mortality over the ensuing 48 h was then determined. Mice that ultimately died became moribund within the first 24 h after challenge; nearly all died within 48 h. As determined by the Reed-Muench method [9], the LD 50 of GBS type III strain M781 was 4 X 104 cfu and the LD go was 2 X 105 cfu. Age-related susceptibility of neonatal mice to GBS injection. Within weeks after birth, human infants appear to rapidly become resistant to infection by GBS. In 'an experiment designed to determine whether mice show a similar age-related susceptibility to infection, 31 mice were challenged with 4 X 104 cfu of strain M781 on days 1, 3, and 5 oflife. Table 1 demonstrates the age-related decrease in susceptibility to infection. One-day-old mice were extremely susceptible, while 5-day-old mice were not killed by this inoculum. Signs of illness (apathy, seizures, anorexia, and pallor of the skin) detected in animals infected within the first 3 days of life were not seen in pups infected on day 5. Recovery of GBS from blood cultures. Twenty neonatal mice were infected with 4 X 104 cfu of strain M781 on day 2 of life; 8 control animals were injected with 0.05 mL of sa-
Table 1. Passive protection of neonatal mice against challenge with type III GBS strain M781. p
Treatment of dams (n)
Survival in pups(%)
vs. PBS
PBS (2) NRS (I) IlI-TT antiserum (2)
2/11 (18) 3/9(33) 22/22 (100)
.62
Neonatal Mouse Model of Group B Streptococcal Infection Ariane K. Rodewald, Andrew B. Onderdonk, Henry B. Warren, and Dennis L. Kasper
Channing Laboratory, Brigham and Women's Hospital, and Division of Infectious Diseases, Beth Israel Hospital. Departments ofMedicine and Pathology. Harvard Medical School. Boston, Massachusetts
Neonatal mice were infected with type III group B streptococcal (GBS) strain M781 by the intraperitoneal route. Age-related susceptibility to challenge was seen within the first 5 days of life. Quantitative blood cultures demonstrated a rapid increase in bacterial numbers during the first 30 h after challenge. Infected pups showed clinical signs of septicemia, and most succumbed within 48 h of challenge. Histopathologic evaluation of the neonates showed bacterial infection within 1 day after challenge. Pregnant adult mice were given a single inoculation of serum raised in rabbits against a tetanus toxoid-conjugated type III GBS polysaccharide vaccine. This serum passively protected 100% of the offspring. This neonatal mouse model of GBS infection and protection may be suitable for study of various forms of intervention.
Received 9 January 1992; revised 6 April 1992. Grant support: National Institutes of Health (AI-23339, AI-30628). Reprints or correspondence: Dr. Ariane Rodewald. Channing Laboratory. 180 Longwood Ave., Boston, MA 02115-5899. The Journal of Infectious Diseases
1992;166:635-9
© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0030$01.00
pathologic and microbiologic features of the experimental disease, as well as the passive protection of neonatal mice after G BS challenge.
Materials and Methods Bacterial strain. GBS type III, strain M781, was originally isolated from a human neonate with GBS meningitis and donated to the Channing Laboratory by C. Baker (Baylor College of Medicine, Houston). Preparation of inoculum. Two or three colonies of strain M781 from a 12-h tryptic soy agar plate (Scott Laboratories, Fiskeville, RI) were inoculated into 10 mL of Todd-Hewitt broth (Difco, Detroit) and grown at 37°C overnight. This starter inoculum was then placed into 250 mL of Todd-Hewitt broth and incubated in a shaker waterbath at 37°C with monitoring of the optical density (OD) at 450 nm until the culture had reached mid-log phase. Bacterial suspensions were diluted to the appropriate viable cell density. on the basis of the OD at 450 nm, as calibrated by previous studies in Todd-Hewitt broth [6]. Mice. Timed-pregnant, CD-I virus antibody-free female mice (Charles River Laboratories, Wilmington, MA) were used for all experiments at 15 or 16 days of gestation. Animals were housed I per cage and received Lab Chow (Purina, St. Louis) and water ad libitum. Mice were watched closely to determine the date of parturition. Blood cultures. Blood was drawn from neonatal pups by percutaneous cardiac puncture with a 27-gauge needle. A 0.02-mL volume of blood was placed into 10 mL of melted (46°C) tryptic soy agar, mixed, and poured into sterile plates. Representative colonies were confirmed as GBS by Gram's stain and CAMP test. Histopathologic examination. Moribund animals were euthanized with diethyl ether and were placed into neutral-buffered 10% formalin for fixation. Paraffin sections were stained with hematoxylin-eosin or with Brown and Hopps stain. Administration ofantiserum to pregnant mice and challenge of neonates. Antiserum was raised in rabbits to the type III GBS
polysaccharide coupled to tetanus toxoid in our laboratory according to previously described methods [7]. This antiserum with an ELISA titer of I:256,000 was administered to pregnant
Downloaded from http://jid.oxfordjournals.org/ at Russian Archive on December 20, 2013
More than 20 years ago, group B streptococci (GBS) were first identified as an important cause of neonatal sepsis and meningitis in humans. The incidence ofGBS infection is 2-3 cases/I 000 live births, with a mortality of r - 20% for infected neonates. Between 25% and 50% of infants surviving neonatal infection continue to show serious neurologic sequelae. Despite improved methods for the early detection of GBS infection and the availability of appropriate antimicrobial agents for treating this disease, a principal issue remains one of prevention. Two-thirds of the cases of GBS disease in the United States are due to capsular type III strains. A correlation has been shown between low levels of maternal antibody to the type III polysaccharide and GBS infection in neonates [l]. Test systems in a number of animals, including adult mice [2, 3], newborn rats [4], and chick embryos [5], have been used in studies of the host immune response to GBS. The use of adult mice for these studies has been problematic, especially when disease is induced with type III GBS, because of lack of virulence in older animals. These in vivo test systems have proven to be either expensive and cumbersome or inadequate in providing end points consistent with the disease as it occurs in human neonates. Although the neonatal rat model more closely simulates human disease, difficulty has been encountered in studying the details of immunity in any species other than the mouse because of the lack ofimmunologic reagents. We have therefore established an animal model system in newborn mice as a practical and inexpensive tool for evaluating both the pathogenesis of the disease and the host immune response. Here we describe the histo-
636
Concise Communications
Results Establishment ofa lethality-dose relationship. To develop a model in mice that simulates GBS septicemia and meningitis in humans, a dose-response experiment was done and the lethal dose of GBS for l-day-old mice (day of birth) was established. Mice of this age were selected to simulate the stage of development at which human neonates with the early-onset form of the disease most frequently become infected. Twenty-four animals were inoculated intranasally with GBS strain M781 at doses of 105-108 cfu in 0.05 mL with no signs of illness. Eighty-three newborn mice received 0.1 mL of broth containing an inoculum ranging from 102 to 108 cfu by the ip route. Mortality over the ensuing 48 h was then determined. Mice that ultimately died became moribund within the first 24 h after challenge; nearly all died within 48 h. As determined by the Reed-Muench method [9], the LD 50 of GBS type III strain M781 was 4 X 104 cfu and the LD go was 2 X 105 cfu. Age-related susceptibility of neonatal mice to GBS injection. Within weeks after birth, human infants appear to rapidly become resistant to infection by GBS. In 'an experiment designed to determine whether mice show a similar age-related susceptibility to infection, 31 mice were challenged with 4 X 104 cfu of strain M781 on days 1, 3, and 5 oflife. Table 1 demonstrates the age-related decrease in susceptibility to infection. One-day-old mice were extremely susceptible, while 5-day-old mice were not killed by this inoculum. Signs of illness (apathy, seizures, anorexia, and pallor of the skin) detected in animals infected within the first 3 days of life were not seen in pups infected on day 5. Recovery of GBS from blood cultures. Twenty neonatal mice were infected with 4 X 104 cfu of strain M781 on day 2 of life; 8 control animals were injected with 0.05 mL of sa-
Table 1. Passive protection of neonatal mice against challenge with type III GBS strain M781. p
Treatment of dams (n)
Survival in pups(%)
vs. PBS
PBS (2) NRS (I) IlI-TT antiserum (2)
2/11 (18) 3/9(33) 22/22 (100)
.62
