Formalin-killed Cells Research Articles

Antibody-independent protection against Pseudomonas aeruginosa infection in mice after treatment with a homologous strain vaccine.

Formalin-killed cells of Pseudomonas aeruginosa strain M-24 elicited an antibody-independent protective effect against P. aeruginosa infection in mice. The effect was observed as early as 6 h after administration and 100% protection was obtained by 48 h. The protective effect could not be attributed to the production of specific antibody. In M-24-treated mice, the bacteria in the peritoneal cavity, blood and liver were eliminated 12 h after P. aeruginosa infection. This suggested that the protective effect was due to enhanced bacterial elimination. The percentage of macrophages in the peritoneal cavity was increased after M-24 administration. Furthermore, the enhanced bacterial elimination was abrogated by treatment of mice with 60Co-irradiation or carrageenan. These findings suggest the involvement of macrophages in the enhanced bacterial elimination observed. The chemiluminescence of peritoneal exudate cells from M-24-treated mice was markedly increased when compared with that of cells from untreated mice. The ability to kill P. aeruginosa in vitro was also greater in macrophages from mice treated with killed M-24 than in cells from proteose-peptone-treated mice. The M-24-treated mice showed enhanced nonspecific protection against infection with lethal doses of P. aeruginosa, Escherichia coli or Listeria monocytogenes. However, susceptibility to LPS in mice was not increased by M-24 treatment. These results suggest that macrophage activation without increasing LPS susceptibility was responsible for the antibody-independent protection induced by killed M-24.

Immune response of ayu against Vibrio anguillarum lipopolysaccharide.

In order to investigate immune response of ayu Plecoglossus altivelis against Vibrio anguillarum lipopolysaccharide (LPS) by intraperitoneal injection, antibody production at various water tem-peratures and amount of the crude LPS that induce protective immunity were examined. Agglu-tinating antibody titer against formalin-killed cell and passive hemagglutinating antibody titer against crude LPS and pure LPS increased 1 week after immunization in the fish reared at 25°C and 3 weeks after the immunization in the fish reared at 15-20°C. The fish reared at 10°C showed lower titers at 5 weeks after immunization. Distinctly higher agglutinating antibody titer against formalin-killed cell was observed in the serum and mucus of the fish immunized with 0.05-0.50 mg of crude LPS than that of the control fish. Those immunized fish showed 100% survival rate after challenge by water born infection, while the control fish showed 13.3 %. From these results, it is concluded that ayu shows good immune response against V. anguillarum LPS at a range of 15-25°C and 0.05 mg of LPS per fish induces an enough immune protection against the infection.

Ｐａｓｔｅｕｒｅｌｌａ ｐｉｓｃｉｃｉｄａリボゾームワクチンによるブリの浸漬免疫

To develop a practical vaccine against pseudotuberculosis, a ribosomal P fraction (RBP) prepared from Pasteurella piscicida was applied to yellowtail Seriola quinqueradiata by immersion method and the immunogenic effects were examined.Group RBP×1 was immersed once with RBP, whereas group RBP×2 was immersed twice with RBP. Agglutination antibody titer, alternative complement pathway activity (ACH50) and phagocytic index were monitored. Artificial challenge was carried out four weeks after immersion. The agglutination antibody titer against formalin-killed cells did not rise significantly in any groups. ACH50 in the RBP×1 and RBP×2 were lower than that in the control. Phagocytic indexes in all the groups were similar. Percentage survival seven days after artificial challenge were: 11%; control, 0%; RBP×l and 0%; RBP×2. The results suggest that RBP is possibly not effective against pseudotuberculosis in yellowtail by immersion method. Furthermore, the immune response of yellowtail seemed to be suppressed by the immersion vaccination with RBP.

Hypersensitivity pneumonitis toParacoccidioides brasiliensisantigens in mice

Hypersensitivity pneumonitis was induced in mice immunized with Paracoccidioides brasiliensis and challenged, one week later, with soluble (SPbAg) or particulate (PPbAg) antigen (formalin-killed yeast cells), administered by the intratracheal route. Between 24 and 48 h post-challenge, animals developed an interstitial and intra-alveolar pneumonitis. Macrophages and lymphocytes arranged focally into loose or mature granulomata were observed by light and electron microscopy. The distribution and fate of antigens was studied by immunofluorescence. Three hours after challenge with SPbAg the lungs showed linear fluorescent deposits, whereas after challenge with PPbAg the pattern was globular, corresponding to the particulate antigen. After 24 and 48 h, the pattern was diffuse and finely granular in both groups, with a decreasing number of animals showing detectable fluorescence. Immunization induced a positive footpad swelling test (FPT) in all animals. After pulmonary challenge, there was a significant decrease in FPT indices, interpreted as desensitization due to trapping of specifically sensitized lymphocytes in the lungs. In conclusion, immunization induced a marked cellular immune response, the inflammatory pattern and the tempo of the induced pneumonitis being compatible with delayed hypersensitivity in the lungs. Immunized mice also cleared the injected antigens rapidly. These data suggest that hypersensitivity pneumonitis may be an expression of pulmonary resistance to infection with P. brasiliensis.

Enzyme-linked immunosorbent assay for detection ofPseudomonas aeruginosa H (Flagellar) antigen

An enzyme-linked immunosorbent assay (ELISA) with goat anti-rabbit IgG conjugated to peroxidase was used to test for the two antigen types of Pseudomonas aeruginosa: b (homogeneous) and a (heterogeneous) which contains the common subantigen (a0) and combinations of subtypes (a, a2 a3 a4). Preparations of b-type flagellar antigen could be distinguished from a-type by using b-adsorbed antisera titers as reciprocals of endpoint dilutions exceeding one million. Extracts from nonflagellated bacteria or purified lipopolysaccharides from the same strain were used as controls, which showed only background activity. Unknown flagellar antigen was determined using both isolated antigen preparations and formalin-killed bacterial cells. The ELISA procedure proved much more sensitive than the slide agglutination procedure: whereas nine of 18 strains tested did not react in the slide agglutination procedure all 18 strains were definitively typed as a or b strains with the ELISA. The ELISA also revealed the presence of a dominant, cross-reacting epitope (a0) in the heterogeneous a-type flagella using either isolated antigen or intact cells.

魚類における感染およびその防御機構に関する研究 ＶＩＩ アユの体表粘液および血清中の抗体の精製と特性

Immunoglobulin(Ig)in the skin mucus of ayu was purified and some biochemical charac-teristics of the purified Ig were compared with those of the purified serum Ig of ayu. Formalin-killed cells of Vibrio anguillarum emulsified with Freund's complete adjuvant was administered to ayu by intraperitoneal injection. Three weeks after immunization, the skinmucus was collected by scraping off the fish body surface with plastic blade. The serum was obtained from the blood taken by severing the caudal peduncle. The Igs in the skin mucus and the serum were purified separately by salting-out with 50% saturated ammonium sulfate, gel filtration on Sephacryl S-300 and ion exchange chromatography on DEAF-Toyopearl 650M. The agglutinating antibody titers of the mucus and the serum were 1:16 and 1:64, respec-tively. Agglutinating activities of both Igs were observed in the fractions right after the void volume from the column of Sephacryl S-300. The Igs from the skin mucus and the serum were eluted at about 0.07M NaCl and at 0.05-0.09M NaCl, respectively, in the linear gradient of NaCl on DEAE-Toyopear1650M. After rechromatography on Sephacryl S-300, both purified Igs gave a single band at the same position on polyacrylamide gel electrophoresis. These purified Igs lost their agglutinating activities by 2-ME treatment and the molecular weights of these Igs were estimated to be about 900, 000 on gel filtration. From these results, it was concluded that the Igs purified from both the skin mucus and the serum of immunized ayu are indistinguishable in biochemical characteristics, and that they are immunoglobulins of IgM type with high molecular weights.

Chemotactic and leukocytosis-inducing activities of eel complement.

Biological activities of eel complement activated through the alternative pathway, namely, chemotactic activity and leukocytosis-inducing activity, were investigated.The normal eel sera were mixed with zymosan or formalin-killed cells of selected bacteria (Escherichia coli, Edwardsiella tarda or Vibrio anguillarum) and incubated at 25°C for 60 min. The sera were separated from zymosan or bacterial cells by centrifugation. And then, this treated sera were heated at 50°C for 30 min.Chemotactic activity of the treated sera were determined by the modified Boyden chamber method. The distance of eel neutrophil migration toward the treated sera were significantly different from that toward the inactivated sera.The peripheral neutrophils increased in number rapidly 6-9 hr after the intraperitoneal injection with the treated sera. There appeared to be the leukocytosis-inducing activity in the treated sera.These biological activites of eel complement indicated in this study may play an important role in the non-specific defence mechanism of eel.

Location of Pasteurella piscicida antigens in tissues of yellowtail Seriola quinqueradiata vaccinated by immersion.

The fate of Pasteurella piscicida antigens in yellowtail Seriola quinqueradiata delivered by immersion exposure to formalin-killed cells, sonicated cells and crude lipopolysaccharide (LPS) was studied. The tissue location of the antigen was determined histologically using the fluorescent antibody technique. The fate of these antigens showed almost the same pattern. After exposure, antigens were detected in the skin from 0 to 60 min, in the gills and stomach from 0 to 12 h, in the intestine from 30 min to 3 d, in the heart from 60 min to 6 h and in the liver, spleen and kidney from 6 h to 24 h. After immersion in crude LPS, phagocytic cells containing P. piscicida antigen were observed in the heart wall and lamina propria of the intestine. The results suggested that the main antigen uptake sites occurred in the gills of the fish and the antigen subsequently entered the systemic circulatory system. It was also suggested crude LPS activated the phagocytic cells in the heart and intestine.

Different types of monoclonal antibodies to Ogawa-specific and group-specific antigens of Vibrio cholerae O1

Nine monoclonal antibodies to Ogawa-specific antigenic determinants of Vibrio cholerae O1 and seven monoclonal antibodies to the Ogawa-Inaba common antigenic determinants were obtained. Specificities and reactivities were examined by slide or microdilution agglutination methods, along with enzyme-linked immunosorbent assays and immunoblotting analysis. In both the Ogawa-specific and Ogawa-Inaba common groups, it was revealed that there were two types of antibodies. One type showed strong agglutination with both live cells and heat-killed (100 degrees C for 30 min) cells in the microdilution agglutination test and in the slide agglutination test (type 1), while the other showed rather weak agglutination with heat-killed cells, especially in the slide agglutination test (type 2). Electronic measurement of agglutinates from these antibodies revealed that the size of aggregates varied. In the case of the type 2 antibodies, the size of aggregates obtained with heat-killed cells was smaller than those obtained with Formalin-killed cells. Results of enzyme-linked immunosorbent assays and immunoblotting analysis showed that all 16 antibodies were to the lipopolysaccharide of V. cholerae O1.

Effect of Edwardsiella tarda lipopolysaccharide immunization on phagocytosis in the eel.

The effects of Edwardsiella tarda lipopolysaccharide (LPS) on the immune response of eel were studied by determination of antibody titer and phagocytic activity of the blood and by determina-tion of survival rate to experimental challenge. Crude LPS and formalin killed cell (FKC) of E. tarda were injected twice intramuscularly in the eels. To compare the protective effect of the pre-parations, the eels were challenged with 2.4×107 cells/fish of live E. tarda, strain EF-1. The agglutination antibody titer of both LPS and FKC immunized eels was high when com-pared to the controls. In vitro phagocytic activity of the total blood was high in both LPS and FKC immunization groups than that found in the control group, whereas the higher average ac-tivity was shown in the LPS immunization group. Only the eels immunized with LPS preparation showed survival to the experimental challenge. The results in the eel confirm the capacity of the LPS to increase not only the antibody titer, but also the level of phagocytosis and the protection to the infection.

直接蛍光抗体法によるウナギ主要細菌性疾病の迅速診断

Direct fluorescent antibody technique (FAT) has been carried out for diagnosis of Edwardsiella tarda, Aeromonas hydrophila and atypical Aeromonas salmonicida infections. Fluorescent antibody (FA) was prepared against E. tarda EF-l, A. hydrophila CA-1 and atypical A. salmonicida C-5. To remove cross reactivity between A. hydrophila and atypical A. salmonicida, CA-1 FA and C-5 FA were absorbed with formalin-killed cells of atypical A. salmonicida and A. hydrophila, respec-tively. To determine if cross reaction occurred, the fish pathogen bacterial suspensions were heat fixed to slides and stained with the dilute FA preparations. At field survey, the heat-fixed smears of the kidney and the head ulcerative lesion from diseased eel were stained with FA preparations. In addition, these tissue samples were inoculated on 0.5% NaCl brain heart infusion agar 10% horse serum tryptosoy agar. EF-1 FA, CA-1 FA and C-5 FA reacted only to their respective antigens by dilution or absorption. From the 79 diseased eel, the respective numbers of eel where the bacteria were detected by the method were as follows: E. tarda, 60, 51; A. hydrophila, 15, 15 and A. salmonicida, 10, 7 (bacteria, by FAT, by culture). The FAT diagnosed 8 mixed infections as compared to 5 by culture, These results indicate that FAT was superior to culture method for diagnosis of the bacterial diseases in eel.

Modulation of Bhanja Virus Infection in Mice

Randombred (ICR) and inbred (C57B1/6) 4-wk-old SPF male mice were infected extraneurally with Bhanja virus ( Bunyaviridae) and subjected to various treatments. Immunosuppression with cyclophosphamide (CPA) affected the course of the infection when a higher dose (10 6 suckling mouse intracerebral LD 50) of the virus and 2 or 3 injections of CPA (150 mg/kg each) were given: then a part of the animals died due to viral encephalitis, whereas all the CPA-untreated infected mice survived. A dual peripheral infection with Bhanja and Lipovnik ( Reoviridae) viruses did not cause any symptomatic response, and the host's humoral antibody was slightly stimulated. When Bhanja virus was given prior to, or simultaneously with, tick-borne encephalitis virus ( Flaviviridae), a moderate decrease of the mortality (due to tick-borne encephalitis) occurred. A mixed peripheral infection of mouse with Bhanja virus and Cryptococcus neoformans, did not result in a fatal virus encephalitis of the host, nor was cryptococcosis affected substantially. However, formalin-killed cells of the fungus (“cryptococcin”) administered before the extraneural inoculation of Bhanja virus caused an 8-fold increase of antibodies neutralizing the virus; a mild therapeutic or protective effect of cryptococcin on encephalitis after an intracerebral application of Bhanja virus was also observed.

Immune response of eel to Edwardsiella tarda lipid.

To confirm the properties of the lipid from Edwardsiella tarda as immunosuppressor in the eel, suggested by previous studies, lipid extracted from the whole cell (Lipid), crude lipopolysaccharide (LPS), formalin killed cell (FKC) and whole cell without lipid (CWL) were injected intramuscularly in the eels. To compare the protective effect of the preparations, the eels were challenged with live E. tarda, strain EF-1.The eels immunized with the Lipid preparation did not show differences in protection against challenge in comparison with the control group, and the highest survival rate was recorded in eels immunized with the crude LPS preparation. The antibody titer of the Lipid immunization group was very low and the highest was in the crude LPS immunization group. In vitro phagocytic activity of the total blood was significantly lower in the Lipid immunization group than that found in the control group, whereas higher activity was shown in the crude LPS immunization group. The phagocytic activity of FKC and CWL immunization groups was not significantly different from that of the control group. The results confirm immunogenic properties of E. tarda lipid as immunosuppressor, which decreases the level of phagocytosis in the eel.

Blastogenic responsiveness of spleen cells fromLegionella pneumophila-infected mice immunosuppressed with cyclophosphamide

Although guinea pigs are highly susceptible to experimental infection withLegionella pneumophila, mice are considered resistant. In the present study it was found that, although untreated mice resisted lethal infection with up to 107L. pneumophila, mice treated with three divided doses of cyclophosphamide became 10–100 times more susceptible. Injection of mice with 150 mg cyclophosphamide/kg body weight 96 and 48h prior to and on the same day as intraperitoneal challenge with graded dose ofL. pneumophila resulted in markedly increased lethality. Approximately half of the mice pretreated with cyclophosphamide succumbed to 106 legionellae within 4–10 days after infection, and all treated animals given 107 bacteria died. Legionellae were readily recovered from spleen, lymph nodes, and liver of surviving mice 4–10 days after infection, but not thereafter. Sensitization of mice with Legionella antigen was evident by the lymphocyte blastogenic test in vitro, by use of spleen cells at various times after infection. Mice given graded doses ofL. pneumophila evinced enhanced responsiveness to either formalin-killed whole cell vaccine, cell-free sonicate, or purified outer membrane antigen when tested in vitro on days 3 and 5. Peak responses generally occurred 20–35 days after infection. Mice given none or one dose of cyclophosphamide and injected with legionellae showed enhanced responses on day 5 of culture in vitro, a time when spleen cells from control nonsensitized animals showed much lower responses. Surviving mice given three doses of cyclophosphamide had lower blastogenic responses, generally as low as that occurring with spleen cells from nonsensitized animals. Thus suppression of immune responses of mice by cyclophosphamide substantially increased susceptibility toL. pneumophila and depressed blastogenic responsiveness.

The role of encapsulation in the pathogenesis of anaerobic Gram-positive cocci

The pathogenicity of 22 anaerobic and facultative Gram-positive cocci (AFGPC) was investigated by inoculating them into mice and determining their ability to cause subcutaneous abscesses. Only 11 heavily encapsulated isolates (greater than 50% of the cells were encapsulated) induced abscesses. However, when the other 11 isolates were injected with Bacteroides sp. or facultative and aerobic bacteria, abscesses were formed in 8 of the 11 combinations. The AFGPC recovered from the mixed infections contained many encapsulated cells. Encapsulation also occurred in cocci injected with capsular material or with Formalin-killed cells of Klebsiella pneumoniae or capsule-positive Bacteroides sp. After acquisition of capsules, these strains could induce abscesses on reinoculation in mice.

Changes in glycogen content of neutrophils in eel, Anguilla japonica by bacterial infection.

Glycogen content of neutrophils in the eel which were injected with some extraneous materials such as formalin-killed cells, 2% solution of casein and physiological saline were determined and their locomotion and phagocytosis were also examined. The eel injected with bacteria had the highest content of neutrophil glycogen, ranged from 74.6 μg to 105.9 μg per 107 neutrophils. The values in those injected with 2% solution of casein and physiological saline were 60.1-66.9 μg and about 45 μg, respectively. In the locomotion of the nuetrophils, the neutrophils from the eel injected with 2% solution of casein migrated best, and those from the eel injected with bacteria did in the next place. The bacterial phagocytosis of the neutrophils was examined in vitro in the whole blood. Glycogen-rich neutrophils showed the most remarkable phagocytosis, though the locomotion was not promoted.

Characteristics of extracellular products of Flavobacterium sp., a pathogen of bacterial gill disease.

The extracellular products (ECP) of Flavobacterium sp. were studied. The ECP were obtained from the strain BGD 7721. (ATCC 35035) cultured on CGY agar. Hemagglutinating activity of the ECP for erythrocytes from several kinds of animals and agglutinating activity for formalin-killed bacterial cells (BGD 7721) were detected. By immersing juvenile rainbow trout in the solution, the ECP induced clubbing of gill filaments and, then, fusing of lamella, which were characteristics of BGD. Protease, phosphatase and phosphoamidase were detected in the ECP, but hemolysin and endotoxin were not. The serological analysis indicated that the ECP had two types of antigen, one specific to itself and the other common to the washed formalin-killed cells.

Protection of mice against Vibrio vulnificus disease by vaccination with surface antigen preparations and anti-surface antigen antisera.

Vaccination of mice with either Formalin-killed cells or cell extracts of a virulent strain and a weakly virulent strain of Vibrio vulnificus or with rabbit antisera (AS) against the Formalin-killed cells and cell extracts protected against the virulent strain of V. vulnificus. However, the virulent strain vaccines and AS elicited a significantly stronger immune response than the weakly virulent strain vaccines and AS. Adsorption of the AS with either the homologous or heterologous Formalin-killed cells significantly reduced the ability of the AS to protect mice. The major protective antigen(s) in the cell extracts migrated in the void volume of Sephacryl S-400 superfine, was not effectively sedimented by centrifugation at 100,000 X g for 2 h, had an isoelectric point of 3.8 to 4.2, and was sensitive to boiling or autoclaving for 15 min, periodate oxidation, and exposure to pH 12 but was resistant to 56 degrees C, trypsin, pronase, RNase, neuraminidase, and pH 4.5. Electron microscopy revealed that the virulent strain possessed a more dense ruthenium red-staining layer on its outer membrane and had a much smoother surface than the weakly virulent strain. Our results provide evidence that a major protective antigen and virulence factor of V. vulnificus is a heat-labile, acidic polysaccharide located on the bacterial surface.

Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: Treatment with intravenous immune globulin

Intravenous immune globulin was used to treat Pseudomonas aeruginosa infected, burned mice. Protection was dose-related and large amounts of intravenous immune globulin administered early in the infection process were necessary for full protection. Intravenous immune globulin is processed differently in unburned, burned, and burned, infected mice with significantly lower plasma levels in the burned, infected animals 36 hours after a standard dose is given. At 30 hours post-treatment, bacterial counts in the skin and liver tissue of untreated, burned, infected mice are significantly higher than in mice given a protective dose of intravenous immune globulin. Adsorbtion of intravenous immune globulin with heat-killed cells reduces its protective effects; adsorbtion with formalin-killed cells reduces its protective effects even more.

Efficacy of direct immersion vaccination of ayu Plecoglossus altivelis with Vibrio anguillarum bacterin.

There was no mortality in ayu (Plecoglossus altivelis) which had mean body weight of 4.2 or 9.2 g during the rearing period after direct immersion in underground water containing lyophilized formalin-killed cells or sonicated cells of Vibrio anguillarum. A high level of protection against vibriosis was found in fish immersed directly in 0.94 or 9.4 (wet) g/l lyophilized or sonicated bacterin. The efficacy was no different for fish with a mean body weight of 4.2 g immersed for 1, 3, or 10 min in 1 (wet) g/l lyophilized bacterin. The duration of protective immunity in fish with a mean body weight of 8.0 g lasted 60 days after direct immersion in 0.94 or 9.4 (wet) g/l of lyophilized bacterin.