カイコの微生物感染症における生体防衛に関する研究

The effect of vaccination on the silkworm larvae, Bombyx mori against per os inoculation of Bacillus thuringiensis spore and crystal (Endotoxin) complex was studied. The 2nd instar larvae of the silkworm were fed daily during the 2nd instar, with inactive B. thuringiensis T 84 Al (ca. 10^5 spores/larva/day) spore and crystal complex which was treated by heat (100℃ for 10 min.) or formalin (2% HCHO for 30 and 60 min.). After the vaccination the larvae were exposed to an active B. thuringiensis spore and crystal complex at the first day of the 3rd instar. No remarkable changes in larval susceptibility against B. thuringiensis were observed between the vaccinated and the control larvae. Also the vaccination of the silkworm larvae during the 3rd or the 2nd to 3rd larval instars showed the same results. Acquired resistance of the silkworm larvae to per os inoculation of B. thuringiensis toxin did not develop under the condition of the present experiment. Pseudomonas aeruginosa caused rapid septisemia of the silkworm by hemocoelic injection. The median lethal dose (bacterial cells/pupa) varied from 2 to 80 in the silkworm pupa. Vaccination with heat (100℃ for 5 min.) or formalin killed (2% HCHO for 60 min. at 37℃) P. aeruginosa or with live vaccine such as E. coli, produced an acquired resistance in the silkworm against P. aeruginosa. Vaccinated silkworm was able to withstand an exposure to large amounts of P. aeruginosa, usually 100 times of the median lethal dose. Bacterial vaccines produced an acquired resistance of the silkworm, however, the injections of egg albumin and physiological saline were completely ineffective for the development of an acquired resistance. Acquired resistance of the silkworm pupa reached its maximum level 12 hrs. after vaccination and maintained high level at least for 5 days. The normal silkworm hemolymph showed slight bactericidal activity against P. aeruginosa and E. coll. Bactericidal titer of the normal hemolymph varied from time to time depending on the rearing condition and the silkworm strain. The bactericidal activity of the silkworm was enhanced by the vaccination. The silkworm hemolymph immunized with formalin killed P. aeruginosa was effective not only against P. aeruginosa but also against E. coli. It was not effective against Staphylococcus aureus. Stability tests of bactericidal activity were carried out using both immunized and normal hemolymph. The bactericidal activity of the hemolymph remained unchanged after dialysis against 0.05 M phosphate buffer pH 7.2. Heat treatment at 70℃ for 15 min. decreased bactericidal activity in the hemolymph. Various kinds of bacterial vaccines had the ability to produce the bactericidal activity in the silkworm hemolymph against P. aeruginosa and E. coli. In many cases the silkworm failed to produce specific bactericidal substance against the bacteria which were used for the vaccination. The bactericidal activity of the immunized hemolymph was significantly decreased by zymozan treatment. The separation of bactericidal substance from treated zymozan was unsuccessful. The bacterididal substance in the immunized hemolymph was fractionated by gel-filtration on sephadex G-100 column chromatography. The column was equilibriated by 0.05 M phosphate buffer pH 7.2 and eluated by the same buffer solution. The fractionated bactericidal substance showed strong bactericidal activity against P. aeruginosa and E. coll. It was slightly effective against Serratia marcescens and Proteus vulgaris but not against S. aureus. A partially purified bactericidal substance was not inactivated at 70℃ for 15 min., or at 56℃ for 30 min. but the treatment with trypsin (100 μg/ml) or M/200 periodic acid destroyed the bactericidal activity. The bacteriolytic activity was observed when the Micrococcus lysodeikticus suspension was mixed with partially purified bactericidal substance. Also the partially purified bactericidal substance reduced the viscosity of glycolchitin and was confirmed as enzymatic action of lysozyme or chitinase. Both the immunized hemolymph and the normal hemolymph of the silkworm showed bacteriolytic activity when the M. lysodeikticus suspension was mixed with these hemolymph, The bacteriolytic activity of the silkworm hemolymph was not increased by vaccination. Apparently the bacteriolytic activity of the hemolymph did not correspond to the bactericidal activity of the immunized hemolymph. The bactericidal activity and the bacteriolytic activity were eluated to the same fraction by gel-filtration on sephadex G-100. It is suggested that the bactericidal substance have similar characteristics of basic protein such as lysozyme. An attempt was made to find viral inhibitory factor in insects. Silkworm larval h emolymph infected with nuclear-polyhedrosis was centrifuged at 105, 000 g for 120 min, and the virus was removed. The surpernatant was precipitated 33.3%, 50%, and 80% saturation with ammonium sulfate adjusted pH 7.0. The precipitate was dissolved in a samll amount of distilled water and dialyzed against 0.05 M phosphate buffer, pH 7.2 in a visking tube (24/32). The dialysate of 33.3% saturation and of 50% saturation, inactivated the nuclearpolyhedrosis virus in vitro at 27℃ for 30 min.. However, the same fraction from healthy larval hemolymph was completely ineffective. A fraction form vaccinated pupal hemolymph injected with formalin treated infected hemolymph, was slightly effective. Terapeautic effect and defensive effect of the viral inhibitory factor were examined in vivo. In defensive test, the silkworm pupae were injected the viral inhibitory factor 12 hrs. and 24 hrs. prior to the infection. In the other experiment, the silkworm pupae were injected with nuclear-polyhedrosis virus of the silkworm and the viral inhibitory factor was injected 8 min. and 120 min. later. The effects of viral inhibitory factor were unsatisfactory in these conditions. The viral inhibitory factor was not inactivated at 50℃ 30 min. or by the treatment with nagarse. However, ether extraction or heat treatment at 70℃ 15 min. decreased viral inhibitory activity. Purification of viral inhibitory factor was carried out by gel-filtration on sephadex G-200. The viral inhibitory activity was found in both fast eluated and slow eluated fractions. Also the purification was performed by column chromatography on DEAE-cellulose. The viral inhibitory factor was absorbed on DEAE-cellulose colum (equilibriated by 0.02 M phosphate buffer, pH 7.2) and was eluated by 0.5 M phosphate buffer, pH 7.2+1M NaCl. The partially purified viral inhibitory factor showed strong viral inhibitory activity but protein content was very low

Studies of an Acquired Resistance on Microbial Infections in the Silkworm (Bombyx mori L.)

The effect of vaccination on the silkworm larvae, Bombyx mori against per os inoculation of Bacillus thuringiensis spore and crystal (Endotoxin) complex was studied. The 2nd instar larvae of the silkworm were fed daily during the 2nd instar, with inactive B. thuringiensis T 84 Al (ca. 10^5 spores/larva/day) spore and crystal complex which was treated by heat (100℃ for 10 min.) or formalin (2% HCHO for 30 and 60 min.). After the vaccination the larvae were exposed to an active B. thuringiensis spore and crystal complex at the first day of the 3rd instar. No remarkable changes in larval susceptibility against B. thuringiensis were observed between the vaccinated and the control larvae. Also the vaccination of the silkworm larvae during the 3rd or the 2nd to 3rd larval instars showed the same results. Acquired resistance of the silkworm larvae to per os inoculation of B. thuringiensis toxin did not develop under the condition of the present experiment. Pseudomonas aeruginosa caused rapid septisemia of the silkworm by hemocoelic injection. The median lethal dose (bacterial cells/pupa) varied from 2 to 80 in the silkworm pupa. Vaccination with heat (100℃ for 5 min.) or formalin killed (2% HCHO for 60 min. at 37℃) P. aeruginosa or with live vaccine such as E. coli, produced an acquired resistance in the silkworm against P. aeruginosa. Vaccinated silkworm was able to withstand an exposure to large amounts of P. aeruginosa, usually 100 times of the median lethal dose. Bacterial vaccines produced an acquired resistance of the silkworm, however, the injections of egg albumin and physiological saline were completely ineffective for the development of an acquired resistance. Acquired resistance of the silkworm pupa reached its maximum level 12 hrs. after vaccination and maintained high level at least for 5 days. The normal silkworm hemolymph showed slight bactericidal activity against P. aeruginosa and E. coll. Bactericidal titer of the normal hemolymph varied from time to time depending on the rearing condition and the silkworm strain. The bactericidal activity of the silkworm was enhanced by the vaccination. The silkworm hemolymph immunized with formalin killed P. aeruginosa was effective not only against P. aeruginosa but also against E. coli. It was not effective against Staphylococcus aureus. Stability tests of bactericidal activity were carried out using both immunized and normal hemolymph. The bactericidal activity of the hemolymph remained unchanged after dialysis against 0.05 M phosphate buffer pH 7.2. Heat treatment at 70℃ for 15 min. decreased bactericidal activity in the hemolymph. Various kinds of bacterial vaccines had the ability to produce the bactericidal activity in the silkworm hemolymph against P. aeruginosa and E. coli. In many cases the silkworm failed to produce specific bactericidal substance against the bacteria which were used for the vaccination. The bactericidal activity of the immunized hemolymph was significantly decreased by zymozan treatment. The separation of bactericidal substance from treated zymozan was unsuccessful. The bacterididal substance in the immunized hemolymph was fractionated by gel-filtration on sephadex G-100 column chromatography. The column was equilibriated by 0.05 M phosphate buffer pH 7.2 and eluated by the same buffer solution. The fractionated bactericidal substance showed strong bactericidal activity against P. aeruginosa and E. coll. It was slightly effective against Serratia marcescens and Proteus vulgaris but not against S. aureus. A partially purified bactericidal substance was not inactivated at 70℃ for 15 min., or at 56℃ for 30 min. but the treatment with trypsin (100 μg/ml) or M/200 periodic acid destroyed the bactericidal activity. The bacteriolytic activity was observed when the Micrococcus lysodeikticus suspension was mixed with partially purified bactericidal substance. Also the partially purified bactericidal substance reduced the viscosity of glycolchitin and was confirmed as enzymatic action of lysozyme or chitinase. Both the immunized hemolymph and the normal hemolymph of the silkworm showed bacteriolytic activity when the M. lysodeikticus suspension was mixed with these hemolymph, The bacteriolytic activity of the silkworm hemolymph was not increased by vaccination. Apparently the bacteriolytic activity of the hemolymph did not correspond to the bactericidal activity of the immunized hemolymph. The bactericidal activity and the bacteriolytic activity were eluated to the same fraction by gel-filtration on sephadex G-100. It is suggested that the bactericidal substance have similar characteristics of basic protein such as lysozyme. An attempt was made to find viral inhibitory factor in insects. Silkworm larval h emolymph infected with nuclear-polyhedrosis was centrifuged at 105, 000 g for 120 min, and the virus was removed. The surpernatant was precipitated 33.3%, 50%, and 80% saturation with ammonium sulfate adjusted pH 7.0. The precipitate was dissolved in a samll amount of distilled water and dialyzed against 0.05 M phosphate buffer, pH 7.2 in a visking tube (24/32). The dialysate of 33.3% saturation and of 50% saturation, inactivated the nuclearpolyhedrosis virus in vitro at 27℃ for 30 min.. However, the same fraction from healthy larval hemolymph was completely ineffective. A fraction form vaccinated pupal hemolymph injected with formalin treated infected hemolymph, was slightly effective. Terapeautic effect and defensive effect of the viral inhibitory factor were examined in vivo. In defensive test, the silkworm pupae were injected the viral inhibitory factor 12 hrs. and 24 hrs. prior to the infection. In the other experiment, the silkworm pupae were injected with nuclear-polyhedrosis virus of the silkworm and the viral inhibitory factor was injected 8 min. and 120 min. later. The effects of viral inhibitory factor were unsatisfactory in these conditions. The viral inhibitory factor was not inactivated at 50℃ 30 min. or by the treatment with nagarse. However, ether extraction or heat treatment at 70℃ 15 min. decreased viral inhibitory activity. Purification of viral inhibitory factor was carried out by gel-filtration on sephadex G-200. The viral inhibitory activity was found in both fast eluated and slow eluated fractions. Also the purification was performed by column chromatography on DEAE-cellulose. The viral inhibitory factor was absorbed on DEAE-cellulose colum (equilibriated by 0.02 M phosphate buffer, pH 7.2) and was eluated by 0.5 M phosphate buffer, pH 7.2+1M NaCl. The partially purified viral inhibitory factor showed strong viral inhibitory activity but protein content was very low