Antigenic relationships among thermophilic actinomycetes.

Antigens from Micropolyspora faeni, Saccharomonospora viridis, Thermoactinomyces candidus, T. sacchari and T. vulgaris were prepared by growing them on dialysate of trypticase soy broth. Sera from rabbits immunized with these antigens were used to study cross-reactivity between thermophilic actinomycetes by antigen-antibody crossed immunoelectrophoresis and by agar gel double diffusion. Mi. faeni and S. viridis showed some degree of cross-reaction, but both failed to show any cross reactivity with Thermoactinomyces species. Antigens from Thermoactinomyces cross-reacted with members within the genus, but no reactivity against S. viridis or M. faeni antisera was detected. Hence, the presence of antibodies to several thermophilic actinomycetes in the sera of patients may be attributed to the exposure of the individual to different thermophilic actinomycetes rather than to the antigenic cross-reactivity between the organisms.     

Micropolyspora faeni causes airway inflammation but not hyperresponsiveness in sensitized ponies

We assessed the effect of aerosol Micropolyspora faeni challenge in two groups of ponies by measuring lung function, airway reactivity to aerosol histamine, and bronchoalveolar lavage fluid cytology. One group of ponies was sensitized by subcutaneous injection of M. faeni in complete Freund's adjuvant, and the other group served as control. In both groups of ponies, measurements were made at base line and 5 h after aerosol administration of 30 ml of saline or 30 ml of 1% wt/vol particulate M. faeni antigen in saline. Saline challenge had no effect on any of the measured variables. M. faeni challenge had no effect on pulmonary mechanics or gas exchange in the control group but significantly increased respiratory frequency and minute ventilation and decreased arterial CO2 tension in the sensitized ponies. In both groups of ponies, aerosol M. faeni challenge significantly increased total white blood cell count and neutrophil numbers in bronchoalveolar lavage fluid while large mononuclear cell numbers decreased. Airway responsiveness was unaltered by saline or M. faeni challenge in both pony groups. We conclude that aerosol M. faeni challenge induces pulmonary neutrophilia and abnormalities of ventilation but is not accompanied by airway hyperresponsiveness in sensitized ponies.     

Isolation and partial characterization of a genus common antigen and species specific antigen of Capnocytophaga

Sixteen strains of Capnocytophaga were isolated from the pocket of a localized juvenile periodontitis patient. These strains were divided into four groups on the basis of morphological and physiological traits. Strains from group I and group III were identified as C. ochracea and group II as C. sputigena. An antigen common to genus Capnocytophaga was purified utilizing immunoabsorbent chromatography from lysates obtained by sodium dodecyl sulfate treatment of C. ochracea strain S1. An antigen specific to C. ochracea was prepared by sequential gel filtration and preparative isoelectric focusing. The genus common and species specific antigens isolated were immunologically unique and pure when tested by immunoelectrophoresis and immunodiffusion against rabbit antisera prepared to Capnocytophaga and other gram-negative rods. The genus common antigen was susceptible to trypsin and pronase digestion, was soluble in chloroform-methanol, but was unaltered by ribonuclease and deoxyribonuclease treatments and periodate oxidation. Antigenicity of the species specific antigen was destroyed by periodate oxidation. The genus common antigen appeared to be lipid-associated protein, while the species specific antigen consisted mainly of carbohydrate. These specific immunological reagents would be valuable in diagnosing and monitoring diseases.     

Micropolyspora faeni antigens of three commercial extracts

The protein composition of three commercial extracts of Micropolyspora faeni, produced in U.S.A., England and Italy has been evaluated by agarose gel electrophoresis.By crossed immunoelectrophoresis, tandem-crossed immunoelectrophoresis and by a modification of this last technique, the antigenic composition and the common antigens of the extracts have been investigated. Hyperimmune rabbit serum and a pool of five human sera with precipitins to Micropolyspora faeni have been used as source of antibody.Different quantity and quality of protein content was observed in the available batches. Different antigenic composition was also observed, not directly related to the different proteins contained therein; three antigens were definitely common to all extracts and two of them represented the major antigens of each extract.Despite the total protein content, the major and common antigens were found in similar concentrations in all three products examined. Therefore, the discrepancies observed in the precipitin reactions using the three commercial Micropolyspora faeni extracts are due to differences in the minor antigen composition of the extracts.     

Cloning of Brucella genes in Escherichia coli K12 cells and analysis of products of the cloned genes]

The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.     

Analysis of Antigens in Mycobacterium Paratuberculosis

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good. Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations. Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity. The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

绿僵菌属血清学的初步研究

本文采用凝集试验和免疫电泳的方法对不同来源的七株绿僵菌属真菌进行了免疫学对比研究。抗原是孢子悬液和菌丝体清液,通过对家兔接种抗原而获得抗血清。每一种抗原与其同源抗血清和异源抗血清进行交叉试验,并对凝集试验的结果以及免疫电泳反应产生的沉淀弧数目和免疫电泳图谱加以对比分析,试验结果表明:不同菌株间存在着明显的抗原类似性,同时各菌株间也表现出一定的抗原专一性。根据试验数据对供试菌株进行血清学分型的结果与形态学分类相符合。     

Antigenic variability of Aspergillus fumigatus strains.

The effect of culture media, temperature of incubation, and continuous shaking of cultures, on the reactivity and yield of antigens of Aspergillus fumigatus were evaluated. It was found that AOAC medium was superior to Czapek medium and shake cultures yielded better results than stationary cultures. However, antigens from stationary cultures in AOAC medium incubated at 30 degrees C for 3 weeks were equally as good as antigens obtained from 2-week-old shake cultures. Antigens from 11 selected strains of Aspergillus fumigatus were used to test antibody activity in 33 sera from patients with various forms of aspergillosis and 35 normal controls by the agar gel double-diffusion method. The results showed that the reactivity of individual antigens varied from 42 to 87%, indicating that antigens from more than one strain of Aspergillus fumigatus may be used. The cross-reactivity between strains were studied by two-dimensional immunoelectrophoresis. The use of polyacrylamide gel electrophoresis and crossed immunoelectrophoresis in the quality assurance of Aspergillus antigens is discussed.     

Isolation anc characterization of actinophages of Thermoactinomyces and Micropolyspora

Two new actionphages infecting thermophilic actinomycetes were isolated from lysogenic cultures of Thermoactinomyces candidus and Micropolyspora faeni. The physiochemical properties, morphology, host range, electron microscopy, and immunology of the two phages, phi-115A (ATCC 29680-B1) and phi-150A (ATCC 29681-B1), are reported.     

Serological Comparison of Two Races of Fusarium oxysporum f. sp. lupini

Water-soluble mycelial antigens from two physiological races (2 and 3) of Fusarium oxysporum L. sp. lupini were compared by tandem-crossed immunoelectrophoresis. When antiserum against race 3 was tested some 50 antigens were detected. The two races had apparently almost identical antigenic patterns differing only in one antigen specific to race 3. This specific antigen might be related to the virulence of this fungus.     

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

Soluble and membrane-bound enzyme-active antigens of rat-liver lysosomes.

Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent-solubilized membrane fractions were analysed in two-dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non-specific esterase, arylsulphatase, glycosidases and L-leucyl-beta-naphthylamidase. When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme-active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non-identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly. Closer analysis of these results indicated that several seemingly identical enzyme-active proteins occurred both in soluble and membrane-associated forms. However, while many of the membrane antigens expressed 2-4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane-associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri-, di- and monophosphatase activities. L-Leucyl-beta-naphthylamidase activity was found in one additional acid-phosphatase-active precipitate.     

Serological characterization of a ribonuclease from Hordeum vulgare

Specific rabbit antisera against purified Hordeum vulgare seedling RNase I from two winter barley cultivars each formed a single precipitin band when reacted with the homologous crude tissue extract. RNase antigen from either cultivar was equally reactive with both antisera when evaluated by immunodiffusion and immunoelectrophoresis. A small but consistent difference in anti-RNase specificity between cultivars was shown by passive hemagglutination inhibition, suggesting that molecular differences may exist between the two RNase antigens. Immunodiffusion and rocket immunoelectrophoresis were used to qualitatively test the cross-reactivity of protein preparations from various members of the genus Hordeum and species from other related grass genera. Neither antiserum showed cross-reactivity with soluble protein preparation from species outside the genus Hordeum. A few species within the genus Hordeum were cross-reactive. A modification of rocket immunoelectrophoresis was developed to determine the amount of RNase in unpurified tissue extracts. The technique involved a template-reservoir which allowed detection of 250 ng RNase in tissue extract volumes of 50 μl. The amount of RNase in unpurified protein extracts from the two cultivars of barley was similar.     

Serological characteristics within the genus Desulfovibrio

Antisera were prepared against one strain each of Desulfovibrio desulfuricans, D. vulgaris and D. salexigens. The antisera were tested for cross reactivity against 36 heterologous Desulfovibrio strains by both agglutination titration and by double immunodiffusion precipitin plates.Generally no cross-reaction was demonstrated by agglutination even between heterologous strains of the same species, suggesting that the surface antigens of Desulfovibrio are highly specific. In immunodiffusion plates a single apparently genus-specific surface antigen could be shown to be present in all but two of the strains tested. Although other common precipitin bands showed the presence of some antigens common between heterologous strains these appeared to be randomly distributed among the strains tested, with the exception of one band shown to be generally specific to strains of D. salexigens. With this exception no other precipitin band could be shown to be consistently specific to any other species, nor consistently common to more than one species.     

Serological Studies of Clostridium botulinum Type E and Related Organisms II. Serology of Spores

Pure spore antigens for the immunization of rabbits were prepared by enzymic digestion of vegetative components and separation of the cleaned spores in polyethylene glycol. Spore antisera were prepared to strains representative of toxigenic Clostridium botulinum type E; nontoxigenic boticin E-producing variants; nontoxigenic nonproducers of boticin E; nontoxigenic "atypical" strains, which differ somewhat from C. botulinum type E in their physiology; C. botulinum types A and B; and C. bifermentans. They were tested against these and additional strains representative of the above groups, other types of C. botulinum, and other Clostridium species. There was no evidence of agglutination of flagellar or somatic antigens of vegetative cells by these antisera. Agglutination and agglutinin absorption tests showed common antigens among toxigenic type E strains and nontoxigenic variants, both producers and nonproducers of boticin E. Some nontoxigenic "atypical" strains varied in their ability to be agglutinated by type E antisera, and others did not agglutinate at all. Of those atypical strains that were not agglutinated, one was agglutinated by C. bifermentans antiserum. Antisera prepared against C. botulinum types A and B and C. bifermentans did not agglutinate the spores of type E or its variants nor share antigens common to each other. Similarly, antisera to type E, its nontoxigenic variants, and nontoxigenic atypical strains did not agglutinate other C. botulinum types or any other Clostridium species investigated.     

Evidence for a Multiplicity of Capsular Types Among Staphylococcus aureus Strains

The Smith diffuse variant and the wound mucoid strain of Staphylococcus aureus were shown to exhibit serologically distinct capsules. The Welwood and K-6 strains of S. aureus were tested to determine their capsular types. Both Welwood and K-6 were found to be representative of the Smith capsular type. An additional 13 isolates of S. aureus from mice were tested. Gel double-diffusion tests and immunoelectrophoresis of staphylococcal antigens disclosed the possible existence of at least two additional capsular types. Passive hemagglutination tests carried out with cells sensitized with 1 mg of antigen per ml showed a multiplicity of cross-reacting antigens. However, cells sensitized either with 0.1 or 0.05 mg of antigen per ml and reacted with antisera absorbed with 10 or 1 mug/ml showed the presence of a specific antigen in each strain of S. aureus. Corroborative evidence for a multiplicity of capsular types was obtained by the specific capsular reaction. At least four capsular types of S. aureus were found. The prototypic strains for these antigens are the RLM or wound strain, the Smith diffuse strain, and mouse strains designated 36T and 43R. We propose to designate these types 1, 2, 3, and 4, respectively.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.     

Identification of Isolates of Clostridium perfringens Types C and D by Agglutination and Fluorescent-Antibody Methods

Agglutination and fluorescent-antibody methods were employed for screening Clostridium perfringens types C and D from 393 isolates of this organism. All of 50 strains which were isolated in Japan and were agglutinable with an antiserum prepared against a stock strain of type C no. 3182 toxigenically belonged to type C, but the antiserum showed no cross-agglutination with any of type C strains isolated in Denmark. All of the latter strains, however, were agglutinated by an antiserum prepared against a Danish strain, CWC11. Of 64 strains, showing heat-labile agglutinability by type D antiserum L9, 22 strains were toxigenically identified as type D strains which can be divided into three groups by the heat-stable antigens; no strains which were L-agglutination-positive but O-agglutination-negative were epislon-toxigenic. All of 13 strains, the heat-stable antigen of which was agglutinable by a type D antiserum VX81, were toxigenically type D strains. The results of fluorescent-antibody tests were almost in agreement with those of agglutination test with type C strains and completely with those of the O-agglutination test with type D strains. No beta-, epsilon- or delta-toxigenicity could be demonstrated in strains which were not agglutinated by our test sera for types C and D strains. Further examination of cultural properties of Japanese and Danish type C strains revealed that the two groups were considerably different in urease production, capsule formation, and delta- and alpha-toxigenicities.     

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.