Protective effects of clostridium sordellii LT and HT toxoids against challenge with spores in guinea pigs.

The protective effects of Clostridium sordellii lethal toxin (LT) and hemorrhagic toxin (HT) toxoids against challenge with spores in guinea pigs were investigated. Purified LT and partially purified HT were obtained from the culture supernatant of C. sordellii strain 3703, and then were treated with formalin to make toxoids. LT. HT and combined LT and HT (LT/HT) toxoid vaccines were prepared by mixing each toxoid with an aluminum phosphate gel as adjuvant. Guinea pigs immunized twice with the respective toxoid vaccines were challenged with spores of strains 3703 or KZ1047. The latter strain does not produce HT. LT toxoid vaccine conferred protection against challenge with strain KZ1047, but not strain 3703, in guinea pigs. All guinea pigs immunized with HT toxoid vaccine died after challenge with spores of either strain. LT/HT toxoid vaccine gave complete protection against challenge with spores of strains 3703 and KZ1047 to guinea pigs. These results suggest that not only LT toxoid, but also HT toxoid, are essential protective antigens of C. sordellii.

Quantitative diversity of 67 kda protective antigen among serovar 2 strains of Erysipelothrix rhusiopathiae and its implication in protective immune response.

Mouse monoclonal antibodies (MAbs), raised against the NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain Kyoto (serovar 2), recognized two different epitopes on a single protein of molecular weight 67 kDa. The MAbs were classified as protective or non-protective against strain Fujisawa (serovar 1). In immunoblotting analysis using the MAbs, fifteen wild strains were shown to contain different amounts of 67 kDa protective antigen. Each formalin-killed whole cell vaccine (bacterin) prepared from the fifteen wild strains conferred different levels of protection against strain Fujisawa in mice. Bacterins prepared from wild strains with larger amounts of 67 kDa protective antigen tended to give high levels of antigen-specific antibody and better protection to mice. These results indicate that the amount of 67 kDa protective antigen which influences the induction of protective immune responses may vary substantially among the strains of E. rhusiopathiae (serovar 2).

The protective effect of Clostridium novyi type B alpha-toxoid against challenge with spores in guinea pigs.

Clostridium novyi (C. novyi) Type B alpha-toxin was purified from culture supernatant by column chromatography, and was inactivated by formalin. A purified alpha-toxoid vaccine was prepared by mixing it with an aluminum phosphate gel adjuvant. Guinea pigs immunized twice with 4 micrograms or more of alpha-toxin survived against challenge with C. novyi Type B spores. Anti-alpha-toxin (antitoxin) titer was measured by toxin neutralization test using Vero cells. All of the guinea pigs having antitoxin titers of 10 units (U) or more at challenge were survived. In another experiment, guinea pigs were immunized with crude alpha-toxoid vaccines prepared by inactivated culture supernatant or by adding broken bacterial cells to the former. In this experiment, 10 U of antitoxin titer was the border of survival or death after challenge. Guinea pigs with antitoxin titers of less than 5 U, 5 U and 10 U died at 2, 3 to 4 and 4 days, respectively, after challenge. These results suggest that C. novyi alpha-toxin was the main protective antigen against challenge exposure to spores in guinea pigs.

Protective effect of NaOH-extracted Erysipelothrix rhusiopathiae vaccine in pigs.

A vaccine was prepared from a NaOH-extracted antigen of the Kyoto strain (serovar 2) of Erysipelothrix rhusiopathiae (E. rhusiopathiae) with an oil adjuvant, and was injected twice at 3-week intervals into SPF pigs and conventional pigs with maternal antibodies. After the second vaccination, IgG-GA titers of immunized SPF pigs were more than 256-fold at 3 weeks, and immunized pigs with maternal antibodies were 64-fold at 7 weeks. The pig with maternal antibodies vaccinated once with live vaccine had less than 4-fold titers. The ELISA antibody titers which were measured by using the NaOH-extracted antigen showed similar transition to the IgG-GA antibody titers. All immunized pigs and nonvaccinated control pigs were challenged with the strains Fujisawa (serovar 1a) or Saitama-1 (serovar 2). After challenge exposure, all pigs immunized with the NaOH-extracted vaccine showed no clinical signs and survived, and the pig immunized with the live vaccine had a local rhomboidal lesion at the site of the injection. Nonvaccinated pigs developed typical symptoms of E. rhusiopathiae infection and one of them died. After the autopsy, the challenge strains were not recovered from the main organs except tonsils of the pigs immunized with the NaOH-extracted vaccine. These results indicated that the NaOH-extracted vaccine induces a protective effect in pigs with maternal antibodies as well as in SPF pigs negative for such antibodies, and that 67-64, 62-60 kDa proteins in the NaOH-extracted antigen play an important role in protecting against E. rhusiopathiae infection.

A field trial of oil adjuvanten trivalent Actinobacillus pleuropneumoniae vaccine.

The trivalent vaccine of A. pleuropneumoniae serotype 1, 2 and 5 (AP3V) was prepared in the oil-in-water type adjuvanten form. At an SPF farm, the vaccinated pigs were observed for their antibody response, finishing rate, and lung lesions at the time of slaughter and for injection scars. The CF titers against serotype 1, 2 and 5 started to rise after the second injection, showed the highest titer at 30 days after injection and then gradually decreased in vaccinated pigs. The finishing rate in the vaccinated group was 91.6% and that in the control group immunized with commercial vaccine was 60%. The lungs in the control pigs showed severe pneumonia with hyperemia, pleural adhesion and abscess. In contrast, vaccinated pigs showed slight pneumonia. Injection scars were not observed in vaccinated pigs 100 days after the second injection. In conclusion, the pigs immunized with AP3V were sufficiently protected against A. pleuropneumoniae infection and the trial proved to be satisfactory in the safety of the vaccine under field conditions.

Protective effect of the combined vaccine prepared from cell-free-antigen of Actinobacillus pleuropneumoniae serotypes 1, 2 and 5 in pigs.

Cell-free-antigens prepared from a concentrated culture supernatant of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) serotypes 1, 2 and 5 were mixed and emulsified with oil adjuvant. The combined vaccine of these 3 serotypes of A. pleuropneumoniae was tested for its ability to confer protection. Pigs immunized with the combined vaccine survived and showed no clinical signs against an intratracheal challenge with A. pleuropneumoniae. In contrast, control pigs inoculated with concentrated culture media emulsified with oil adjuvant developed typical symptoms of pleuropneumonia after challenge inoculation.

Turkey rhinotracheitis virus isolated from broiler chicken with swollen head syndrome in Japan.

Turkey rhinotracheitis (TRT) virus was first isolated from a commercial broiler chicken with swollen head syndrome (SHS) in Japan. At the same time, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), avian reovirus (ARV), Escherichia coli (E.coli), Morganella morganii, and Proteus mirabilis were also isolated from the same broiler chicken. The presence of antibodies to TRT virus was confirmed in the sera of 34-day-old chickens of the flock with SHS, however the antibodies to TRT virus were undetectable in the sera of 17-day-old chickens. In this investigation, we confirmed avian pneumovirus infection in chickens in Japan, and the virus and other agents may be considered as a cause of SHS.

Protective efficacy of cell-free-antigen of Actinobacillus pleuropneumoniae in mice.

Cell-free-antigen (CFA) vaccines of strain Y-1 (serotype 1), G-4 (serotype 2) and E-3 (serotype 5) of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) were prepared by emulsifying concentrated culture supernatant with oil-adjuvant. Mice immunized with the CFA vaccine had a high survival rate (90-100%) against challenge with the homologous strain. They also had cross-protective activity against challenge with the heterogeneous strains but their survival rate was low (20-50%). On the other hand, mice immunized with whole cell vaccine showed serotype specific protection and only a little cross protection. The protective antigens of the CFA were investigated. MAbs were produced by the standard method using spleen cells of mice immunized with CFA. MAbs to Apx I, II, III and capsular antigen of serotype 5 were obtained. Only MAbs to Apx I showed hemolysin neutralization activity among them. The protective effect of these MAbs against A. pleuropneumoniae infection were examined by passive immunization. Administration of Apx I MAb to mice extended survival time after challenge with serotype 5. The mice showed partial cross-protection against challenge with serotype 1. Survival rate was considerably low after the challenge infection. None of the mice given MAbs to Apx II or III were protected against challenge with serotype 5. The mice given MAb to capsular antigen of serotype 5 had a high survival rate (70%) against a challenge with a homologous serotype. Furthermore, mice given MAbs against Apx I and capsular antigen of serotype 5 were completely protected against a challenge with A. pleuropneumoniae serotype 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenicity of condensed pyridazines with different substituents.

A total of 24 compounds were prepared by introducing an N-oxide, a hydrazino group, a methoxy group or a chloro group into 3 kinds of condensed pyridazines: pyrido[3,4-d]pyridazines, pyrido[2,3-d]pyridazines and phthalazines. The mutagenicity of these 24 compounds was assessed by the Ames method using two tester strains (Salmonella typhimurium TA98 and TA100). No mutagenic activity was detected with any of the 3 condensed pyridazines without substituents or any of the 5 condensed pyridazines with a methoxy group. The compounds with N-oxide in the pyridazine ring showed no or only very weak mutagenicity. However, when an oxide was introduced into the nitrogen of the pyridine ring, the mutagenicity against strain TA98 was higher than that of any other test compound. All compounds with a hydrazino group were mutagenic against strains TA98 and TA100, irrespective of the presence or absence of S9 mix-induced metabolic activation. 1-Hydrazinophthalazine (hydralazine) which has been clinically used as an antihypertensive agent was weakly mutagenic. The introduction of a chloro group increased the bactericidal effects of the condensed pyridazines, thus hampering the assessment of mutagenicity. A majority of the compounds which were found to be mutagenic in this study required no metabolic activation with S9 mix.

Passive protection of mice against Actinobacillus pleuropneumoniae infection by monoclonal antibodies.

Mouse monoclonal antibodies (MAbs), Y-4b, F-6b and K-6a recognized as capsular antigens of A. pleuropneumoniae serotypes 1, 2 and 5, respectively, and MAb H-1b as lipopolysaccharide (LPS) of serotype 2 were produced. We examined the passive immunizing efficacy of these MAbs on A. pleuropneumoniae infection in C3H/HeJ mice. On the challenge infection of homologous serotype strains, they showed a sufficient protective effect in immunized mice. It was concluded that MAbs recognized as capsular antigen and LPS have a serotype-specific protective effect on A. pleuropneumoniae infection, suggesting the important role in preventing A. pleuropneumoniae infection.

AS-183, a novel inhibitor of acyl-CoA: cholesterol acyltransferase produced by Scedosporium sp. SPC-15549.

A novel compound, AS-183, which inhibits acyl-CoA: cholesterol acyltransferase (ACAT), was isolated from the culture broth of a fungus, Scedosporium sp. SPC-15549. AS-183 inhibited ACAT activity in an enzyme assay system using rabbit liver microsomes with an IC50 value of 0.94 microM. AS-183 also inhibited cholesterol ester formation in HepG2, CaCo2, and THP-1 cells with IC50 values of 18.1, 25.5, and 34.5 microM, respectively.