Fig 1.
Kinetics of bacterial colonization of B. canis RM6/66 in mice.
Animals were divided into 4 groups (n = 30) consisting of lower dose (105, 107), high dose (109), or control (PBS) groups. Animals were inoculated i.p. and 5 animals from each group were euthanized at 1-, 2-, 4-, 6-, 9-, and 12-weeks post-infection. Colonization was evaluated in the liver (A), spleen (B), uterus (C), bone marrow (D), lung (E), and kidney (F). Bacterial recovery is displayed as the total CFU/g. Data points represent the mean recovery per gram of tissue plus the standard deviation for all animals in each dose group at each time point. Statistical significance was determined by ANOVA followed by Tukey’s multiple comparisons test. One asterisk, P <0 0.05. Two asterisks, P < 0.01. Three asterisks, P < 0.0001.
Fig 2.
Splenic weight in mice inoculated with B. canis RM6/66 or PBS over 12 weeks.
Splenomegaly was induced by a high dose (109) of B. canis by 1-week post-infection (A). Splenic weight peaked at 2-weeks post-infection and then declined with no significant differences noted between treatment groups and the control group by 6-weeks post-infection. Data points represent the mean spleen weight plus the standard deviation for all animals in each dose group at each time point. Mean spleen weight from each dose group (n = 5) was compared at each time point to mean spleen weight of the uninfected control mice (n = 6) and statistical significance was determined by ANOVA followed by Tukey’s multiple comparison test. Three asterisks, P <0.0001.
Fig 3.
Histopathology of the spleen in mice infected with B. canis.
(A) Representative images of histopathology and immunohistochemistry of the spleen of mice infected intraperitoneally with PBS (top row) or B. canis RM6/66 at a low dose (105, second row), mid-range dose (107, third row), or high dose (109, bottom row) at 2-weeks post-infection. (B) Sections were scored for severity of histiocytic inflammation from 1–4 (S1 Table) at 2 weeks post-infection, and mean scores were compared using ANOVA followed by Tukey’s multiple comparisons test. (C) Sections were scored using the same system at 12 weeks post-infection. The black circles in the left panel indicate the section highlighted to higher magnification in the middle panel. Infection with B. canis resulted in infiltration by epithelioid macrophages within the marginal zone and lymphoid follicles (*). Brucella antigen was detected within the cytoplasm of macrophages by immunohistochemistry (arrows). Magnification 4x (left column, HE, scale bar = 100 μm), 20x (middle column, HE, scale bar = 50 μm), 40x (right column, IHC with DAB chromagen, bar = 20 μm). Two asterisks, P<0.01, four asterisks, P <0.0001.
Fig 4.
Histopathology of the mesenteric lymph nodes in mice infected with B. canis.
(A) Representative images of histopathology and immunohistochemistry of the mesenteric lymph nodes of mice infected intraperitoneally with PBS (top row) or B. canis RM6/66 at a low dose (105, second row), mid-range dose (107, third row), or high dose (109, bottom row) at 2-weeks post-infection. (B) Sections were scored for severity of histiocytic inflammation from 1–4 (S1 Table) at 2 weeks post-infection, and mean scores were compared using ANOVA followed by Tukey’s multiple comparisons test. (C) Sections were scored using the same system at 12 weeks post-infection. The black circles in the left panel indicate the section highlighted to higher magnification in the middle panel. Infection with B. canis resulted in infiltration by epithelioid macrophages within the medullary sinuses zone and surrounding lymphoid follicles (*). Brucella antigen was detected within the cytoplasm of macrophages by immunohistochemistry (arrows). Magnification 4x (left column, HE, scale bar = 100 μm), 20x (middle column, HE, scale bar = 50 μm), 40x (right column, IHC with DAB chromagen, bar = 20 μm). Three asterisks, P<0.001, four asterisks, P <0.0001.
Fig 5.
Histopathology of the liver in mice infected with B. canis.
(A) Representative images of histopathology and immunohistochemistry of the liver of mice infected intraperitoneally with PBS (top row) or B. canis RM6/66 at a low dose (105, second row), mid-range dose (107, third row), or high dose (109, bottom row) at 2-weeks post-infection. (B) Sections were scored for severity of lesions on a scale of 1–4 based on the number of microgranulomas and prevalence of periportal inflammation (S1 Table) at 2 weeks post-infection, and mean scores were compared using ANOVA followed by Tukey’s multiple comparisons test. (C) Sections were scored using the same system at 12 weeks post-infection. The black circles in the left panel indicate the section highlighted to higher magnification in the middle panel. Infection with B. canis resulted in multifocal, random microgranuloma formation (*) and predominantly mononuclear periportal inflammation. Brucella antigen was detected within the cytoplasm of macrophages in the microgranulomas by immunohistochemistry (arrows). Magnification 4x (left column, HE, scale bar = 100 μm), 20x (middle column, HE, scale bar = 50 μm), 40x (right column, IHC with DAB chromagen, bar = 20 μm). Four asterisks, P <0.0001.
Fig 6.
Histopathology of the uterus in mice infected with B. canis.
Representative images of histopathology and immunohistochemistry of the uterus of mice infected intraperitoneally with PBS (top row) or B. canis RM6/66 at a low dose (105, second row), mid-range dose (107, third row), or high dose (109, bottom row) at 2-weeks post-infection. The black circles in the left panel indicate the section highlighted to higher magnification in the middle panel. Infection with B. canis resulted in histiocytic, neutrophilic, and lymphocytic peritonitis and metritis (*). Brucella antigen was detected within the cytoplasm of macrophages in the outer myometrium by immunohistochemistry (arrows). Magnification 4x (left column, HE, scale bar = 100 μm), 20x (middle column, HE, scale bar = 50 μm), 40x (right column, IHC with DAB chromagen, bar = 20 μm).
Fig 7.
Humoral response to B. canis infection in mice.
Anti-Brucella specific IgG indirect ELISA was detected in sera from mice infected intraperitoneally with PBS or B. canis at doses of 105, 107, and 109 at 1, 2, 4, 6, 9, and 12-weeks post-infection. Mice in all treatment groups developed a significantly higher IgG response compared with the uninfected control group. Total Brucella-specific IgG is expressed as the mean absorbance per group plus the standard deviation. Statistical significance was determined by ANOVA followed by Tukey’s multiple comparison of each treatment group (n = 5) to the uninfected controls (n = 6). Three asterisks, P <0.0001.