Protective efficacy and safety of Brucella melitensis 16MΔmucR against intraperitoneal and aerosol challenge in BALB/c mice

Abstract

Brucellosis is a zoonosis of nearly worldwide distribution. Vaccination against this pathogen is an important control strategy to prevent the disease. Currently licensed vaccine strains used in animals are unacceptable for human use due to undesirable side effects and modest protection. Substantial progress has been made during the past 10 years toward the development of improved vaccines for brucellosis. In part, this has been achieved by the identification and characterization of live attenuated mutants that are safer in the host but still can stimulate an adequate immune response. In the present study, the identification and characterization of the mucR mutant (BMEI 1364) as a vaccine candidate for brucellosis was conducted. BALB/c mice were vaccinated intraperitoneally at a dose of 10(5) CFU with the mutant to evaluate safety and protective efficacy against intraperitoneal and aerosol challenge. All animals vaccinated with the vaccine candidate demonstrated a statistically significant degree of protection against both intraperitoneal and aerosol challenge. Safety was revealed by the absence of Brucella associated pathological changes, including splenomegaly, hepatomegaly, or granulomatous disease. These results suggest that the 16MΔmucR vaccine is safe, elicits a strong protective immunity, and should be considered as a promising vaccine candidate for human use.

Fig. 1.

Kinetics of clearance of 16MΔmucR from mice. Forty 6- to 8-week-old female BALB/c mice were used to evaluate the persistence and replication of 16MΔmucR. Mice were inoculated intraperitoneally with either (i) 10⁶ CFU in 100 μl of 16MΔmucR or (ii) 10⁶ CFU in 100 μl of the parental strain 16M. Groups of four mice were euthanized via carbon dioxide asphyxiation at 1, 3, 6, 9, or 12 weeks postinfection. At each time point, the spleens were harvested, weighed, and homogenized in 1 ml of peptone saline. Serial dilutions were prepared, and 100-μl aliquots of each dilution (including the undiluted organ) were plated in duplicate on TSA plates. (A) The levels of infection were expressed as means ± the SEM of individual log CFU/spleen values. (B) The spleen weights were measured and used to compare the mutant strain to the wild-type organism. Statistical significance is based upon a Student t test comparing the deletion mutant to the wild-type strain. The solid line at 0.69 logs represents the lower limit of detection (≥5 CFU).

Fig. 2.

Protection against homologous intraperitoneal 16M challenge. Groups of five female BALB/c mice were vaccinated with 16MΔmucR at either 10⁶ CFU/mouse or 10⁵ CFU/mouse or left unvaccinated as naive controls. At 20 weeks postvaccination all animals were challenged with 6 × 10⁵ CFU/mouse intraperitoneally. At 1 week postchallenge, mice were euthanized via CO₂ asphyxiation, and the spleens, livers, and lungs were collected. The data are reported as the log₁₀ recovery of Brucella from organs. The solid line at 0.69 logs represents the lower limit of detection (≥5 CFU). Statistical analysis was performed by ANOVA for each organ separately, followed by a Tukey's HSD post test comparing all groups to one another.

Fig. 3.

Protection against homologous aerosol 16M challenge. Groups of five female BALB/c mice were vaccinated with 16MΔmucR at 10⁵ CFU/mouse or left unvaccinated as naive controls. At 20 weeks postvaccination, all of the animals were challenged with an aerosol chamber dose of 5 × 10⁹ CFU of 16M/ml. At 4 weeks postchallenge, the mice were euthanized via CO₂ asphyxiation, and the spleens, livers, and lungs were collected. The data are reported as the log₁₀ recovery of Brucella from organs. The solid line at 0.69 logs represents the lower limit of detection (<5 CFU). Statistical analysis was performed by using a Student t test comparing vaccinated to nonvaccinated mice for each organ separately.

Fig. 4.

Histological changes of organs associated with vaccination and challenge. BALB/c mice were vaccinated with 16MΔmucR at 10⁵ CFU/mouse or left unvaccinated as naive controls. At 20 weeks postvaccination, all of the animals were challenged with an aerosol chamber dose of 5 × 10⁹ CFU of 16M/ml. At 2 weeks postchallenge, tissues were collected for histology. The histology of the nonvaccinated but challenged animals (A, B, and C) were compared to vaccinated but challenged animals (D, E, and F). The lungs (A and D), livers (B and E), and spleens (C and F) were compared to determine the reduction in pathology afforded by vaccination with the 16MΔmucR mutant. Naive mice are depicted (G, H, and I) for comparison. Hematoxylin and eosin staining was used (×10 magnification).