Vaccination of chickens with liposomal inactivated avian pathogenic Escherichia coli (APEC) vaccine by eye drop or coarse spray administration.

One hundred and twenty 30-day-old specific-pathogen-free chickens were inoculated with the liposomal inactivated avian pathogenic Escherichia coli (APEC) vaccine by eye drop or coarse spraying. All of the chickens produced anti-lipopolysaccharide antibodies of the IgG subclass in their sera as well as IgA antibodies in their oral mucus. The results demonstrated a rise in antibodies in the serum of chickens administered the APEC vaccine through nonparenteral mucosal routes. Bacterial counts in the blood decreased, and clinical signs were moderated in the vaccinated chickens after challenge with a strain of APEC. No harmful effects from the vaccination were observed. The liposomal inactivated APEC vaccine described in this paper would contribute to a practical method of control for avian colibacillosis.

Influence of antigenic forms and adjuvants on protection against a lethal infection of Aujeszky's disease virus.

The influence of antigenic forms and adjuvant types on protection against a lethal infection of Aujeszky's disease virus (ADV) in mice was investigated. Antiviral IgG2a antibody response against particulate (inactivated ADV) and soluble antigen (ADV solubilized with deoxychorate-Na) in approximate order of extent was ISA70>QS-21>positively charged liposome>negatively charged liposome>weak negatively charged liposome>ISA25>lablabside F saponin>aluminum phosphate gel>non adjuvant. Particulate antigen induced higher IgG2a antibody production than soluble antigen. Particulate antigen combined with ISA70, ISA25 or positively charged liposome gave 100, 50 and 40% protection to mice, respectively. In contrast, soluble antigen plus ISA70 conferred 30% protection on mice. Immunogens using the other adjuvants gave

Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants.

Adjuvant and haemolytic activities of 47 saponins purified from medicinal and food plants were examined. The compounds showed various levels of both adjuvant and haemolytic activities. Soyasaponins and lablabosides showed strong adjuvant activity but little haemolytic activity. Jujubosides showed strong adjuvant and haemolytic activities. Escins showed weaker adjuvant activity than the adjuvant-control, but strong haemolytic activity. Comparison of the functional groups of each saponin revealed that the acyl residue in saponin, the aldehyde group at carbon 4 in aglycone, and branched sugar chains attached to aglycone, were not essential for adjuvant activity. Furthermore, saponins with an acyl residue or oxide-ring moiety tended to show haemolytic activity. These results suggest that the adjuvant activity of saponins does not relate with haemolytic activity. It is considered that not only the functional groups themselves, but the overall conformation harmoniously consisting of such functional groups, affects adjuvant activity of saponins.

Characterization of flagellin from Clostridium chauvoei.

Differential centrifugation and cesium chloride-equilibrium centrifugation were used to purify the flagella from the strain Okinawa of the formalin-fixed Clostridium chauvoei. SDS-PAGE profile of purified flagella showed that a major protein band with a molecular mass of 46 kDa, corresponding to the flagellin monomer, and at least two minor protein bands with molecular masses of approximately 73 and 100 kDa were found. The amino acid composition of C. chauvoei flagellin was similar to the flagellin of Salmonella typhimurium and Bacillus subtilis. In addition, C. chauvoei flagellin monomer shared limited sequence homology with the N-terminal amino acid sequence reported for other bacterial flagellins. N-terminal sequences of two minor bands corresponded to the flagellin monomer, indicating that higher molecular mass bands were polymeric forms of the flagellin monomer.

Influence of antigenic forms and adjuvants on the IgG subclass antibody response to Aujeszky's disease virus in mice.

The influence of antigenic forms of Aujeszky's disease virus (ADV) and adjuvant types on the production of IgG subclass antibodies in mice was investigated. Particulate antigen, inactivated ADV, alone induced IgG1 and lower IgG2a antibody production, while the antigen adsorbed onto aluminum phosphate gel (alum) enhanced IgG1 antibody production but suppressed IgG2a antibody production as well as solubilized ADV antigen adsorbed onto alum. QS21 saponin purified from Quillaja saponaria promoted the production of IgG1 and IgG2a antibodies in a large extent against the both particulate and soluble antigens, while this saponin has strong hemolytic activity. Lablaboside F saponin isolated from Dolichos lablab without hemolytic activity, also induced the production of large IgG1 and little IgG2a antibody against both antigens. Oil-based adjuvant, ISA70 of water-in-oil type and ISA25 of oil-in-water type, increased IgG1 and IgG2a antibodies against the both soluble and particulate antigens, whereas a combination of ISA25 and soluble antigen reduced IgG2a antibody response. These results indicate that IgG1 antibody production was not suppressed by a combination of antigenic form and adjuvant type, however, IgG2a antibody production was influenced.

Influence of cell surface glycoprotein gC produced by pseudorabies virus on cytopathic effect.

The wild-type pseudorabies virus (WT-PRV) produced a round-type cytopathic effect (CPE) in PK-15 cell line of porcine kidney origin, while PRVgCs lacking in gC-transmembrane-anchor region and PRVgC-defecting in gC gene produced a syncytium-type CPE. The mouse embryo cell line (BALB/3T3 clone A31) were transfected with recombinant plasmid of pcDNA3 which incorporated with gC gene. The transfected A31/gC cells were stably expressing gC. Only a round-type CPE was observed in these cells infected with WT-PRV, while a syncytium-type CPE was observed in the cells infected with each of the PRVgCs and PRVgC-. Any viruses described above induced a syncytium-type CPE in A31/pcDNA cells transfected with a plasmid without gC gene. By WT-PRV infection, PK-15 cells generated about 2- or 8-fold more gC than the A31/gC and A31/pcDNA cells when gC was measured by hemagglutination test. Flowcytometric analysis revealed that amount of gC on the cell surface of A31/gC and PK-15 cells increased after infection with WT-PRV. Round-type CPE was observed with the increase of gC. These results suggest that the type of CPE formation induced by PRV is dominated by the amount of gC on the infected cell surface.

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.

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)

Protective effect by cell-free antigen obtained from culture supernatant of phase I Bordetella bronchiseptica.

The cell-free antigen (CFA), with highly hemagglutination activity, obtained from the culture supernatant of Bordetella bronchiseptica was compounded with oil adjuvant to make a component vaccine (CFAV). In the immunization trial in mice, the offsprings whose mothers were immunized with CFAV escaped from death when challenged intrapleurally with virulent strain of B. bronchiseptica. The protective indices (difference of LD50 dose of the challenge strain between immunized and control groups) of the offsprings from CFAV-immunized mothers were over 3.0 in common logarithm value. Moreover, about 90% of the offsprings from CFAV-immunized mothers were negative in nasal turbinate atrophy, while over 80% of them from non-immunized mothers showed obvious turbinate atrophy when challenged intranasally with virulent strain. On the one hand, remarkable differences in the number of bacteria recovered from nostrils were observed between both test groups. It was concluded that CFAV is a very effective vaccine against B. bronchiseptica infection in animals.

Characterization of haemagglutinin from Bordetella bronchiseptica.

In comparison with haemagglutinin (HA)-active strains of Bordetella bronchiseptica, the HA-deficient strains lacked a 150 kDa protein band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Adsorption of partially purified HA with bovine erythrocytes showed the loss of the 150 kDa band in the supernatant. This 150 kDa protein was purified by high-performance liquid chromatography using gel filtration columns. Electron microscopic examination revealed that the purified HA possessed a fine filamentous structure with dimensions of approximately 2 x 150 nm, which was considered to represent the filamentous haemagglutinin of B. bronchiseptica. Both the cell-free antigen obtained from the culture supernatant of phase-I strain of the bacteria and the bovine erythrocytes, which combined with crude HA showed an excellent protective activity of the challenge by virulent strain of B. bronchiseptica in mice.

[Studies on immunity to Babesia gibsoni in dogs immunostimulation by Bordetella bronchiseptica].

Four species of bacteria, Corynebacterium anaerobium 578, Actinobacillus pleuropneumoniae G-4, Mycobacterium bovis BCG, and Bordetella bronchiseptica A-2, were injected intravenously into mice (5 weeks old, ICR-SPF). The clearance of carbon from the blood stream and the weights of the spleen and liver were determined as indicators of RES stimulation. Mouse footpad reaction was assessed as an indicator of delayed-type hypersensitivity to each species of bacteria. The immuno-stimulative activity of each species of bacteria against bovine serum albumin was monitored by passive hemagglutination assay and the macrophage migration-inhibition test in guinea pigs. Based on the results of the experiments described above, B. bronchiseptica was selected as an immunostimulator (Ims) for immunization trials of the hemo-protozoan parasite, Babesia gibsoni, with inactivated merozoites of B. gibsoni (BgK). Twelve dogs, pointers about 6 months old, were divided into four groups of three dogs each. Group 1 dogs were initially injected with Ims, and later injected with BgK and Ims (BgK+Ims) after a 3-week interval. Group 2 and Group 3 dogs were injected twice, at a 3-week interval, with BgK+Ims and BgK, respectively, and Group 4 served as a control. As the results, the serum antibody titres of Group 1 and 2 were several times higher than that of Group 3, and the cell-mediated immunity to parasites was noticeably stimulated by immunization with BgK+Ims. The peak level of parasitemia following the challenge were over 10% for Group 4 and 4.5% for Group 3, while levels for Group 1 and 2 were 2.5% and less than 1%, respectively. No such major clinical signs of babesiosis as jaundice and anemia were observed in Group 1 or 2.

Characterization of a parvovirus isolated from the diarrheic feces of a pig.

A small DNA virus was isolated from the feces of a sow with diarrhea and identified as a parvovirus on the basis of its properties. The virus replicated preferentially in cell cultures of swine origin, including primary porcine thyroid gland and kidney cell cultures in which the cytopathic effect developed. The virus agglutinated erythrocytes of guinea pig, mouse and human group O but not these of chicken. The growth of the virus was inhibited by 5-iodo-2'-deoxyuridine. The virus was resistant to ether and heating at 56 degrees C for 30 min and stable at pH 3.0. The buoyant density of the infectious particles was 1.40 g/ml in CsCl density gradient, and the virions were 27 nm in diameter by electron microscopy. The viral protein seemed to be separated into four polypeptides with molecular weights of 81k, 70k, 66k and 62k daltons respectively. Cross serum neutralization test demonstrated that the virus was antigenically different from porcine parvovirus as well as bovine and canine parvoviruses. These findings and the survey on neutralizing antibody distribution indicated indirectly that another parvovirus which could be antigenically distinguished from well-known porcine parvovirus had been widespread among swine in Japan.