Comparison of the susceptibilities of C57BL/6 and A/J mouse strains to Streptococcus suis serotype 2 infection

Abstract

Streptococcus suis is an important swine and human pathogen. Assessment of susceptibility to S. suis using animal models has been limited to monitoring mortality rates. We recently developed a hematogenous model of S. suis infection in adult CD1 outbred mice to study the in vivo development of an early septic shock-like syndrome that leads to death and a late phase that clearly induces central nervous system damage, including meningitis. In the present study, we compared the severities of septic shock-like syndrome caused by S. suis between adult C57BL/6J (B6) and A/J inbred mice. Clinical parameters, proinflammatory mediators, and bacterial clearance were measured to dissect potential immune factors associated with genetic susceptibility to S. suis infection. Results showed that A/J mice were significantly more susceptible than B6 mice to S. suis infection, especially during the acute septic phase of infection (100% of A/J and 16% of B6 mice died before 24 h postinfection). The greater susceptibility of A/J mice was associated with an exaggerated inflammatory response, as indicated by their higher production of tumor necrosis factor alpha, interleukin-12p40/p70 (IL-12p40/p70), gamma interferon, and IL-1beta, but not with different bacterial loads in the blood. In addition, IL-10 was shown to be responsible, at least in part, for the higher survival in B6 mice. Our findings demonstrate that A/J mice are very susceptible to S. suis infection and provide evidence that the balance between pro- and anti-inflammatory mediators is crucial for host survival during the septic phase.

FIG. 1.

Survival curves of A/J and B6 mice after S. suis infection. Mice were injected i.p. with S. suis serotype 2 (1 × 10⁷ CFU/ml) and mortality was recorded daily for 15 days. S. suis-infected A/J mice showed significantly high mortality during the first 20 h p.i. compared with B6 mice (P < 0.001).

FIG. 2.

Relevant histopathological findings for B6 mice presenting clinical signs of neurological disease or sudden death (day 9 p.i.) after i.p. infection with S. suis. Hematoxylin-eosin staining of brain and heart tissue samples was performed. (A) Micrograph of the brain parenchyma showing an area of malacia and hemorrhages (black arrows). Magnification, ×100. (B) Micrograph of the brain cortex, in which is evident the presence of a bacterial embolus (*) surrounded by neutrophils. Magnification, ×400. (C) Micrograph of the meninges, which are severely and diffusely infiltrated by macrophages and other nonsuppurative inflammatory cells (black arrows). Magnification, ×100. (D) Micrograph of the heart at the atrioventricular valves with a thrombus occluding most of the valvular lumen (black arrows). The thrombus comprises fibrin, inflammatory cells, and bacteria. Magnification, ×200.

FIG. 3.

Bacterial loads in different organs from A/J and B6 mice infected i.p. with S. suis. Bacterial loads are expressed as CFU/ml for blood (A) and as CFU/0.05 g of tissue for the livers (B), spleens (C), and brains (D). Results are expressed as mean (± standard error of the mean [SEM]) results for at least three infected mice per p.i. time point. No significant differences were found between the two mouse strains throughout the experiment (P > 0.05).

FIG. 4.

Kinetics of the expression of inflammatory mediators in A/J and B6 mice infected i.p. with S. suis. (A) TNF-α. (B) IL-12p40. (C) IL-12p70. (D) IFN-γ. (E) IL-1β. (F) IL-6. Cytokine concentrations in sera were assayed by a liquid multiarray system (Luminex), as explained in Materials and Methods. Data are expressed as mean ± SEM (pg/ml). Values for uninfected controls did not show statistically significant changes from 3 h to 72 h. As such, time zero h represents the mean (± SEM) results for noninfected mice throughout the experiment. Asterisks represent significant differences between mouse strains (P < 0.05).

FIG. 5.

Kinetics of serum chemokine expression in A/J and B6 mice infected i.p. with S. suis. (A) KC. (B) MCP-1. (C) RANTES. Chemokine concentration was assayed by a liquid multiarray system (Luminex), as explained in Materials and Methods. Data are expressed as mean ± SEM pg/ml. Time zero h represents the mean (± SEM) results for noninfected mice throughout the experiment.

FIG. 6.

Role of IL-10 in the survival of A/J and B6 mice after S. suis infection. (A) Kinetics of serum IL-10 production in A/J and B6 mice infected i.p. with S. suis. IL-10 concentration was assayed by a liquid multiarray system (Luminex), as explained in Materials and Methods. Data are expressed as mean ± SEM (pg/ml). Time zero h represents the mean (± SEM) results for noninfected mice throughout the experiment. The asterisk represents a significant difference between mouse strains (P < 0.05). (B) Survival of B/6 mice treated with anti-IL-10R MAb (1 mg/ml) prior to challenge with S. suis. Blockage of IL-10R impedes the protective role of IL-10 and thus renders animals significantly more susceptible to S. suis infection. This is evidenced by a course of mortality that is more rapid in treated B6 mice than that in nontreated controls (P = 0.012). (C) Survival of A/J mice treated with rmIL-10 prior to challenge with S. suis. A/J mice were treated with rmIL-10 at different final concentrations and times relative to the infection. It is notable that rmIL-10 treatment has a protective effect, significantly delaying mortality compared with what was observed for the nontreated group (P < 0.001).

FIG. 7.

Effect of blockage of IL-10R on cytokine production. B6 mice were treated with anti-IL-10R MAb (1 mg/ml) before i.p. infection with S. suis serotype 2 (1 × 10⁷ CFU/ml). Serum samples were taken at 24 h p.i., and TNF-α levels measured by an enzyme-linked immunosorbent assay kit (R & D Systems). The asterisk indicates a significant difference between treated and nontreated groups (P < 0.001). Symbols represent values from individual mice, while the horizontal lines indicate the median for each group.