Hiroshi Saito

Role of Pili in the Pathogenesis of Pseudomonas aeruginosa Burn Infection

The present study using three isogenic mutants (F+P−, F−P+, F−P−) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non‐piliated (P−) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non‐flagellated (F−) mutant was at least 105 times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P− bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P− bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P− bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili‐mediated enhancement of virulence of P. aeruginosa was attributed to pili‐mediated enhanced colonization of the organisms at the burned skin surfaces.

Correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of Staphylococcus hyicus

Staphylococcus hyicus was isolated from healthy pigs and pigs affected with exudative epidermitis (EE). Thirty seven strains (P-7 to P-43) were isolated from pigs affected with EE on 8 farms while 131 strains were isolated from healthy pigs bred on 2 farms in Japan. Isolation rate for pigs affected with EE was 100% while that for healthy pigs was 35.4%. The biochemical and cultural characteristics of the isolates from healthy and diseased pigs were identical except for the Voges-Proskauer reaction. The culture supernatant of many isolates caused skin exfoliation in 1-day-old chickens. Therefore, many isolates were considered to produce S. hyicus exfoliative toxin (shET). The rate of shET production by the isolates from piglets affected with EE was 87.5%, while that of the isolates from healthy pigs was 76.1%. shETs were divided in two serotypes by immunodiffusion. Piglets experimentally infected with shET-producing and nonproducing strains were observed. Local skin erythema at the inoculation site was observed with nonproducing strains and disappeared within 48 h, while the skin erythema at the sites inoculated with shET-producing strains did not disappear until 7 days after inoculation. Typical clinical signs, such as exfoliation, exudation and crusting were observed only in the piglets inoculated with shET-producing strains.

Isolation of exfoliative toxin from Staphylococcus hyicus subsp. hyicus and its exfoliative activity in the piglet

Exfoliative toxin was isolated from the sterile cell-free filtrate of 24 h culture of Staphylococcus hyicus subsp. hyicus strain P-1. The partial purification of exfoliative toxin produced by S. hyicus (shET) was performed by precipitation with 50-80% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column and column chromatography on DEAE-cellulose. Partially purified shET (pp-shET) caused exfoliation in piglets at 8 to 12 h after intradermal or subcutaneous injection. However, heat-treated pp-shET did not cause exfoliation in piglets for up to 24 h after injection. On histopathological examination of the skin at 12 h after injection of pp-shET, an intraepidermal cleavage plane was shown between the stratum corneum and stratum granulosum and at the stratum granulosum.

Susceptibility of various animals and cultured cells to exfoliative toxin produced by Staphylococcus hyicus subsp. hyicus

In piglets inoculated with partially purified exfoliative toxin (pp-shET) produced by Staphylococcus hyicus subsp. hyicus, exfoliation was observed at 12 h after injection. Chickens inoculated with the same dose of pp-shET also showed exfoliation within 30 min of injection. However, exfoliation was not demonstrated in mouse, rat, guinea pig, hamster, dog or cat inoculated with pp-shET until 24 h after injection. In cultured cell lines, especially L-929 and Hep-2, the rounding effect occurred after incubation with pp-shET for 1 h. The rounding effect was also seen in five other cultured cells (NCTC 2544, HeLa/S3, HmLu-1, CHO and BHK-21) 6-24 h after exposure to pp-shET. These round cells survived for 72 h after inoculation and formed a monolayer 24 h after changeover to a toxin-free medium. The rounding effect was observed in cells after the formation of the monolayer, but not before. It was suggested that the rounding effect was not caused by the increase in cyclic AMP in cells inoculated with pp-shET but by the cleavage of intracellular contacts.

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.

Preparation and characterization of monoclonal antibodies against bovine B lymphocyte surface antigens.

Two monoclonal antibodies (MoAbs; BLMo-4 and BLMo-10) were prepared by immunizing with a cell line established from peripheral blood mononuclear cells (PBMC) of enzootic bovine leukosis (EBL) cattle. The specificities of these MoAbs were assayed using bovine PBMC. BLMo-4 reacted with all surface immunoglobulin-positive cells (SIg+ cells; B lymphocytes) and also recognized monocytes, but did not react with T lymphocytes. BLMo-10 recognized a majority, although not all, B lymphocytes, but did not react with either T lymphocytes or monocytes. The antigens recognized by BLMo-4 and BLMo-10 were not Ig, Fc or C3 receptors on the surface of B lymphocytes. The reactivity of the MoAbs with mononuclear cells from the lymphoid organs of adult cattle was studied. BLMo-4 and BLMo-10 did not react with any bone marrow cells. BLMo-10 reacted with 7.4% of thymocytes, and stained the medulla of the thymus in the immunoperoxidase assay. In the case of PBMC, spleen and lymph node cells, the percentage of cells positive for BLMo-4 was slightly higher than that of SIg+ cells, but BLMo-10 showed a slightly lower value.

Protective activity and antigenic analysis of fractions of culture filtrates of Erysipelothrix rhusiopathiae

The protective activity in mice and antigenic composition of the culture filtrate of 11 strains of Erysipelothrix rhusiopathiae were compared. Protective activity was found in the first (P-1) fraction obtained by Sephadex G-200 gel filtration of the culture filtrate of each strain. Comparing the 50% protective dose (PD50) of the P-1 fraction of the 11 strains by active immunization, highly protective activity was shown by 5 strains, such as Agata, Fujisawa, Shizuoka-63, Koganei 65-0.15 and SE-9. For the 50% effective dose (ED50) determined by passive protective studies, the four strains, Agata, Fujisawa, Koganei 65-0.15 and SE-9 were shown to be highly protective. However, strains 2179 and 2553 showed low activities in both PD50 and ED50. The highly and weakly protective strains were compared by western blot analysis for the protein components. In most strains tested, there were two protein bands of molecular weight of 64 kDa and 43 kDa. Therefore, these two structural proteins were common to the strains and were associated with stimulation of a protective effect in mice.

Optimum Culture Conditions for Production of Exfoliative Toxin by Staphylococcus hyicus

Optimum culture conditions for the production of exfoliative toxin by Staphylococcus hyicus (shET) were examined. High shET activity was obtained from the culture filtrate of HI and TY broth inoculated with S. hyicus. The pH in these two media ranged from 7 to 8.5 during bacterial culture, while the lowest pH in TS and BHI broth was less than 6. shET activity in the culture filtrate from TY broth inoculated with 107 CFU of S. hyicus per ml was higher than that in TY broth inoculated with 106 and 108 CFU of bacteria per ml. When shET activity in the culture filtrate was measured under various shaking conditions, the culture filtrate shaken at 75 oscillations per min had the highest shET activity of the five shaking conditions. shET activity of the culture filtrate of TY broth to which protease inhibitor had been added was the same as that of TY broth without inhibitor. shET activity in a shaking culture in an Erlenmeyer flask was also the same as that in sac culture and that in shaking culture using a shaking (Sakaguchi) flask. shET activity in TY broth supplemented with 100 mM glucose was significantly lower than that in TY broth without glucose. Based on the above results, the optimum culture conditions for the production of shET were as follows: inoculation of 3 × 109 CFU of S. hyicus strain P‐1 into 300 ml of TY broth in a 2,000‐ml Erlenmeyer flask, and incubation at 37 C with shaking at 75 oscillations per min. Then shET activity of the culture filtrate under appropriate culture conditions was measured after various incubation periods. shET activity was detected 6 hr after inoculation, reached the maximum (253 exfoliative unit/0.1 ml) at 16 hr and decreased between 20 and 48 hr. Thus, the optimum incubation period was determined to be 16 hr. Then the optimum concentration of ammonium sulfate for isolation of shET from the culture filtrate under appropriate culture conditions was examined. The greatest shET activity was obtained from the fraction salted out with 90% saturated ammonium sulfate. Thus, the optimum concentration of ammonium sulfate for the isolation of shET was determined to be 90% saturation.

Preparation of monoclonal antibodies to Theileria sergenti and their reactivity to antigens in experimentally infected cattle

Two distinct monoclonal antibodies (3-H and 11-D) were produced against Theileria sergenti. These two new products, together with monoclonal antibody 1-G obtained in a previous study, were used to detect the parasites in experimentally infected cattle. During the first period of dexamethasone treatment, which was carried out to increase parasitemia in the infected cattle, the number of erythrocytes detected by 3-H, 11-D and 1-D increased in two experimentally infected calves. During the second period of dexamethasone treatment, the number of infected erythrocytes detected by 3-H and 11-D were similarly increased, but the number of infected erythrocytes detected by 1-G did not increase and infected erythrocytes in one calf were not detected by 1-G.