Detection of CPS content of S. suis serotypes in culture.

(A) The growth curve of 05ZYH33 in TSB-FBS culture was monitored through the optical density at 600 nm (OD600) of the culture at the indicated time points. Data are the mean ± SD of three independent experiments (n = 10). (B) The hydrophobicity of 05ZYH33 in the lag, log, and stationary (Sta) phases was measured by BATH assays. A representative photo of the BATH assay (upper panel) and BATH values (right panel). Δ2BSS2, the isogenic strain of 05ZYH33 without CPS; NC, 05ZYH33 suspension without hydrocarbon (n = 4 to 7).

The adherence of 05ZYH33 to epithelial cells and its resistance to phagocytosis

HEp-2 or HBMECs cells were inoculated with 05ZYH33 or Δ2BSS2 in either the log or stationary phase at an infection MOI of 10. (A) Adherence rate of S. suis to HEp-2 cells. (B) Adherence rate of S. suis to HBMECs. (C) S. suis strains in log and stationary phases were incubated with whole blood from naïve mice for 3 h, and the viability of S. suis was determined. Data are from three independent experiments and present as means ± SEM (n = 6 to 16). **P <0.01, ***P <0.001, ****P <0.0001; ns, not significant.

The intranasal infection course in mice.

Mice were intranasally inoculated with 05ZYH33. Total CFUs in various bodily compartments were assessed at the indicated time points after inoculation, (A) NALT, (B) the CSF, (C) the lung, (D) the blood, (E) the spleen, and (F) the heart. The data presented are derived from two independent experiments and presented as means ± SEM (n = 6). N.D., not detected.

The CPS of 05ZYH33 isolated from various bodily compartment following infection.

05ZYH33 was intranasally inoculated in mice. Twelve hour after inoculation, CFUs in NALT, the CSF, and the blood were assessed for (A) hydrophobicity of bacterial cells by BATH assay and (B) CPS by TEM. Arrows indicate the CPS layers. Scale bar, 100 nm. Data in (A) are from two independent experiments and present as means ± SEM (n = 4 to 6). NC, 05ZYH33 suspension without hydrocarbon. ****P <0.0001.

The role of anti-CPS or anti-V5 serum in 05ZYH33 infection.

(A) Schematic illustration of passive transfer of antisera and infection in mice. (B) and (C) Anti-V5 or ant-CPS antiserum was transferred through the i.n. or i.v. route. Six hours later, mice were i.n. challenged with 05ZYH33, then euthanized 12 h post-challenge. CFUs of 05ZYH33 in NALT, the lungs, the blood, and the CSF were determined. Data are the mean ± SEM of three independent experiments (n = 9). *P <0.05, **P <0.01. ***P <0.001.

Detection of 05ZYH33 colonization in the CSF at early time of infection.

(A) Schematic illustration of passive transfer and mouse infection. (B) and (C) Mice were infused with anti-V5 serum through the i.n. route, and inoculated i.n. with 05ZYH33 or Δ2BSS2 6 h later. One hour later, mice were euthanized, and CFUs in NALT, the blood, and the CSF were determined. Data are the mean ± SEM of three independent experiments (n = 10). *P <0.05, **P <0.01, ***P <0.001.

Detection of the presence of 05ZYH33 in the tissue of olfactory nerve area.

Mice were inoculated with acetic acid through the nostril and one hour later infected i.n. with 05ZYH33. (A) Sagittal views of the olfactory system. Sections of the distal nasal cavity and olfactory epithelium from uninfected (a, c, and e) and 05ZYH33-infected (b, d, and f) mice. Red or orange, 05ZYH33; green, the neuronal marker β-tubulin III; blue, DNA. NC, Nasal cavity; OE, olfactory epithelium; LP, lamina propria; CP, cribriform plate; OB, olfactory bulb. (B) One hour or nine days after 05ZYH33 infection, the brain of mice were sectioned for H&E staining. Arrows indicate infiltrated inflammatory cells. (C) and (D) Mice were inoculated with acetic acid or PBS and one hour later infected i.n. with 05ZYH33 or Δ2BSS2. (C) CFUs of 05ZYH33 or (D) Δ2BSS2 in NALT, the CSF, and blood were determined one h after infection.