The role of non-type-specific protein antigens of the cell wall in Streptococcus group A in developing delayed hypersensitivity

The role of the type-nonspecific (TNS) cell-wall antigens of group A streptococci has been determined. The study has been made on guinea pigs sensitized with whole microbial cells or HCl extracts containing TNS antigens. To determine delayed hypersensitivity, the in vitro cytotoxic test on adhering lymph-node cells in the autologous system has been used. The study has shown that sensitization with group A streptococci of different types or with TNS antigens induces the development of delayed hypersensitivity to TNS antigens (or antigen), common for different types of group A streptococci, but specific for this group. HCl extracts containing TNS antigens can be recommended as the preparation for testing delayed hypersensitivity to antigens, specific for group A streptococci.     

Biochemical and immunochemical analyses of the protein components of the cell wall in group-A streptococci isolated by a sparing chemical method]

To study the protein components of the cell wall of group A streptococci, type M 29, a special preparative method was developed (extraction with 1 M hydroxylamine solution, pH 6.0, and subsequent purification). Altogether six protein fractions were obtained. The isolated proteins were found to be a heterogeneous group of molecules, consisting of 25-40 individual proteins with molecular weights ranging between 13 and 94 kD. The study of the protein fractions thus obtained in the immunodiffusion test with rabbit antiserum to the initial protein preparation revealed that these proteins contained type-specific components, 3-6 type-nonspecific protein antigens common with protein antigens of M 1 and M 12, as well as one protein antigen common with type M 1. Fc receptor was shown to be absent. The detected type-nonspecific protein antigens were partially separated by ion-exchange chromatography and some of them could be purified from the admixtures of nucleic acids and group-specific polysaccharide.     

Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).     

Comparative biological characteristics of the L-form of Streptococcus group A and B

The comparative study of the biological and serological properties of the L-forms of streptococci, groups A and B, has been made. Their morphological similarity on the level of light microscopy has been demonstrated. The use of ring precipitation, gel diffusion, passive hemagglutination, aggregate hemagglutination, as well as the immunoferritin technique, has made it possible to establish the presence of specific antigens in the L-forms of streptococci, groups A and B. Serological cross reactions are negligible. The future development of a diagnosticum for the specific indication of these antigens is proposed. The fact of the presence of specific antigens in the L-forms of streptococci in comparison with the initial streptococcal strains has been confirmed.     

Beta haemolytic streptococci of the Lancefield groups E. P and U:Streptococcus infrequens

The physiological and biochemical characteristics of isolates from swine in Sweden and The Netherlands were compared with those of strains from several culture collections. These characteristics were found to be similar for all three Lancefield groups and they form a well defined pattern distinct from other known streptococcal species. It is suggested that these streptococci be classified together in one species:Streptococcus infrequens.Group and type sera of Group E streptococci have no affinity to Group P and Group U streptococci, and vice versa. None of the sera prepared against group E type strains contains the group antibody. Group P and Group U streptococci have an antigen in common. This common antigen is present in formamide extracts. It is not demonstrable in acid extracts. None of the group P sera tested contains the common antibody. Group P serum has to be considered as a type serum. Group U sera contain the common antibody, and when absorbed with group P cells prove to contain another type antibody, which reacts with extracts of most group U strains.Isolates of all three Lancefield groups were obtained from a variety of pathological conditions in swine.     

Localization of group A streptococcal nontype-specific antigens and their occurrence among streptococci of different groups]

It was shown by immunodiffusion methods that nontypespecific antigens revealed in the HCl extracts of streptococcus, group A, were localized in the cell wall. In B, E, H, K, L, M, P, S, T streptococci groups there was revealed only one, and in C and G streptococci groups--two antigens identical to the HTC antigens of streptococci, group A. Besides, an antigen, which was apparently specific specific for group A streptococcus only, was detected. The data obtained should be taken into consideration in the elaboration of improved method of grouping and typing group A streptococcus.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

The Use of Standardized Reference Antigens in the Serogrouping of Streptococci

Streptococci isolated from the dental plaque of five animal species were identified by physiological and serological methods. Twenty-nine strains of streptococci considered to resemble Streptococcus mitior or Strep. bovis on the basis of physiological data reacted with Lancefield group B or group K antisera respectively in tube precipitation tests. Further serological studies with standardized antigens from known serogroup B and K streptococci revealed that only three of these 29 isolates had been serogrouped accurately and carried the appropriate group antigen. Comparisons were made between the reactivity of the antisera produced by Difco and Wellcome Reagents with acid and autoclaved extracts of the strains. It was shown that the accuracy of serogrouping such isolates could be improved if the tests were made in a gel diffusion system that included a reference antigen.     

Heterogenetic antigens of gram-positive bacteria

Chorpenning, Frank W. (The Ohio State University, Columbus), and Matthew C. Dodd. Heterogenetic antigens of gram-positive bacteria. J. Bacteriol. 91:1440-1445. 1966.-Soluble antigens obtained by various methods from gram-positive bacteria were used to modify erythrocytes whose hemagglutinating reactions with immune rabbit sera and normal human sera were then studied. Antigens from all gram-positive organisms studied except corynbacteria altered red cells, causing them to react with specific bacterial antisera and with normal human sera; however, cross-absorption and inhibition tests indicated that at least three different specificites were involved. One of these antigens seemed to be similar to Rantz's streptococcal NSS, which is shared with Staphylococcus aureus and Bacillus spp., and is therefore heterogenetic. Another was found in streptococci but was apparently not present in S. aureus and Bacillus spp. A third antigen, also heterogenetic, appeared to be shared by several species of Bacillus and by S. aureus, but not by streptococci or any gram-negative bacteria. The third antigen was heat-stable at pH 8.0, and appeared to be essentially polysaccharide in nature. Normal human sera varied in their content of antibodies which reacted with erythrocytes modified by extracts from gram-positive bacteria. Whereas some sera reacted very broadly with red cells modified by extracts of practically any gram-positive organism, other sera agglutinated only cells which had been modified by streptococcal antigen.     

Identification of Group B Streptococci by Immunofluorescence Staining

Gamma globulin fractions of rabbit antisera prepared with whole cell vaccines of group B types Ia, Ib, II, and III and labeled with fluorescein isothiocyanate stained group B streptococci type specifically. Type Ic cells, which contain the Ia polysaccharide antigen of type Ia and the Ic protein antigen of type Ib, were specifically stained by both Ia and Ib conjugates. A group B conjugate pool (B pool) that contained one conjugate specific for each group B type at its predetermined titer gave positive fluorescent-antibody (FA) reactions (4+ intensity) with group B stock strains and negative FA reactions (less than 2+ intensity) with stock strains of streptococcal groups A, C through H, and K through U, viridans streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, and representative Enterobacteriaceae. Examination of 883 clinical isolates submitted to the Streptococcus Laboratory (Center for Disease Control, Atlanta, Ga.) for identification revealed a 99.1% agreement between FA and culture-precipitin methods. All 305 group B streptococci identified by culture-precipitin and six nonhemolytic group B streptococci missed initially by culture tests were identified correctly by FA. Results of cultural and FA methods in a double-blind study of 99 vaginal swabs agreed on 96 of 99 strains. Three nonhemolytic group B streptococci were identified first by FA and later confirmed by culture-precipitin tests.     

Immunodiffusion methods of studying the F-fraction antigens obtained from group A streptococci]

F fractions, obtained by the extraction of cultures of group A streptococci with distilled water at different pH, were studied by immunodifusion methods and subjected to chemical analysis. F fractions were shown to contain polyglcerophosphate, antigen E4 and in some cases group polysaccharide. Besides, F fractions were found to contain an antigen insensitive to trypsin and identical to one of the antigens of the thermostable fraction, as well as an antigen sensitive to the action of proteolytic enzymes and common to various types of group A streptococci. The antigen sensitive to the action of proteolytic enzymes were identical to one of the antigens showing no type specificity and contained in HC1 extracts prepared from group A streptococci. In grouping and typing group A streptococci the present of some F fraction antigens unrelated either to polysaccharide or to M substance should be taken into consideration. The antigens of F fraction have no protective properties.     

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.     

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.     

The enhancement of the immunogenicity of streptococcal group-A M protein by conjugation with a synthetic polyelectrolyte]

Immunization with the polypeptide fragment of group A streptococcal protein M conjugated with the copolymer of acrylic acid and N-vinylpyrrolidone in complete Freund's adjuvant has been found to lead to a sharp increase in the level of antibodies to the type-specific determinants of protein M, detected in the enzyme immunoassay (EIA). The possibility of the application of such sera to preliminary typing of streptococci in EIA with the use of whole microbial cells as antigens has been shown. The data on high activity of the sera thus obtained in the bactericidal test with streptococci of the homologous type are presented. Recommendations on the use of sera obtained by the above method for highly precise typing of the virulent cultures of group A streptococci in the bactericidal test are given.     

Determination of epidemiological relationships of Streptococcus agalactiae isolated from bovine mastitis

In the present study 79 streptococcal cultures isolated from subclinical mastitis of 54 cows from seven dairy farms (A-G) in Hesse, Germany, were comparatively investigated using conventional and molecular methods. The isolates could be identified as Streptococcus agalactiae, belonging to Lancefield's serological group B by determination of cultural, biochemical and serological properties and by polymerase chain reaction (PCR)-mediated amplification of species-specific parts of the 16S ribosomal DNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor gene cfb. The investigated group B streptococci were further characterized serologically for specific polysaccharide and protein antigens. Serotyping the isolates revealed a predominance of surface protein antigen X, either alone or in combination with polysaccharide antigen Ia. This could be observed for 39 isolates of farms A, B and C. Six group B streptococci from farm E displayed the serotype pattern III/Rib, two isolates from farm G showed the serotype pattern Ib/calpha. The remaining cultures from farms D and F (n=32) were non-typable. The occurrence of protein Rib could be confirmed by PCR amplification of the gene rib. The two isolates with serotype pattern Ib/calpha also reacted positively for the cbeta-encoding gene bag. Additional properties which allowed a phenotypic characterization of the S. agalactiae were the degree of pigmentation, growth properties in fluid media and soft agar, the surface hydrophobicity, the ability to hemagglutinate rabbit erythrocytes and their resistance reactions to tetracycline and minocycline. The isolates of the seven farms showed identical or almost identical characteristics. The 79 group B streptococci were additionally investigated by macrorestriction analysis of their chromosomal DNA using the restriction endonucleases SmaI, ApaI and SalI. The restriction patterns obtained by pulsed-field gel electrophoresis displayed identical or closely related patterns for the cultures of the various farms. The pheno- and genotypic characteristics of the 79 group B streptococci of the present study revealed that a single S. agalactiae strain or at least closely related subtypes of this strain were responsible for the mastitis situation of the seven farms.     

Cytotoxic effect of lymphocytes on autologous blood monocytes in the presence of streptococcal group A antigens in patients with primary erysipelas

The present work deals with a modification of the cytotoxic test for the determination of the cytotoxic activity of lymphocytes in infectious diseases. This modification is based on the use of the suspension of mononuclear blood cells, simultaneously containing effector cells (sensitized lymphocytes) and target cells (autologous monocytes). The cytotoxic effect on monocytes is observed after the preliminary incubation of nonadhering cells (lymphocytes) with the antigen of microorganisms causing the infectious process. A statistically significant increase in the cytotoxic activity of lymphocytes was recorded in patients with primary erysipelas at the acute period of the disease. The cytotoxic effect has been found to persist at a high level for two weeks. By the end of the disease this effect drops to the level characteristic of clinically normal persons. An elevated level of the cytotoxic activity of lymphocytes in the presence of streptococcal antigens of one type has been detected in 72% of patients with primary erysipelas. This indicates that type-nonspecific streptococcal antigens take part in the formation of delayed hypersensitivity, which is also confirmed by the data obtained in animal experiments.     

Septicaemic infections in pigs,caused by haemolytic streptococci of new Lancefield groups designated R,S and T

Pyogenic streptococci isolated from outbreaks and from sporadic infections in pigs and piglets were characterized by the almost unique combination of the properties of -haemolysis on horse blood agar and acid production from inulin. Three new serological groups were recognized, each with a single antigen different from those of any of the Lancefield groups. These antigens are polysaccharides located in the cell wall. About half the number of haemolytic streptococcal strains isolated from pigs were group R or group S streptococci, a few strains were group T streptococci, and the remaining strains were group L streptococci,S. equisimilis, group E streptococci, or group P streptococci, in this order of frequency. Only one out of about 150 haemolytic strains could not be identified serologically. Group R and group T streptococci differ from group S streptococci by acid production from raffinose. Infections with group R streptococci appeared to occur independently of age, whereas infections with group S streptococci were almost entirely confined to piglets.     

Glutamine synthetase content and immunochemical analysis of different strains of Bordetella pertussis

In this investigation 3 groups of strains isolated from pertussis patients have been studied: typical (group 1), atypical in their cultural properties (group 2), unidentified Gram-negative bacilli agglutinated by pertussis and parapertussis antitoxins (group 3). Besides, B. pertussis cultures, obtained by subculturing 2 museum strains and 2 newly isolated strains on synthetic casein-charcoal agar with subinhibiting doses of antibiotics or specific immune sera added, have been studied. As indicated by the results of this study, strains belonging to groups 1 and 2 contain glutamine synthetase, while in strains of group 3 this enzyme is absent. In immunoelectrophoresis strains of group 3 have been found to contain not a single antigen similar to the antigens of strains belonging to groups 1 and 2. Electrophoresis in polyacrylamide gel has revealed to differences in the protein spectrum of the strains of these 3 groups. The investigation has shown that the determination of glutamine synthetase and immunoelectrophoresis can be used for the differentiation of B. pertussis from similar Gram-negative bacilli. B. pertussis strains, changed as the result of experiments with antibiotics and specific immune sera, have also been shown to retain their antigenic composition and protein spectrum and to have no essential difference in the content of glutamine synthetase.     

Choice of nutrient media for the laboratory diagnosis of Streptococcus group B

The comparative study of a large assortment of liquid and solid culture media used for the cultivation of streptococci in laboratory practice in the USSR and abroad was carried out with the aim of selecting the optimal media for the laboratory diagnosis of group B. streptococci. Liquid media were tested with the use of 7 streptococcal reference strains, and some of these media, found to yield the best results, were selected for tests on clinical material. The use of liquid accumulation media was shown to permit the isolation of group B. streptococcal strains which could not be detected by the direct inoculation of clinical material into dishes with blood agar. The character of hemolysis induced by group B. streptococci in solid media with 5% of blood added was found to depend on the composition of the medium and the conditions of cultivation.     

Immunochemical analysis of water-soluble antigen complexes isolated from typing strains of Pseudomonas aeruginosa of different O-serogroups

Cross immunoelectrophoresis in agarose and immunodiffusion in agar gel were used to carry out the immunochemical analysis of water-soluble antigenic components isolated from P. aeruginosa of different O-serogroups (according to Lanyi's classification). Immunodiffusion revealed the presence of 1--3 common antigens and 1 specific O-antigen in aqueous extracts. Experiments with the use of cross immunoelectrophoresis indicated that 1--12 common antigens could be detected in aqueous extracts. The reference preparation, produced on the basis of the cell mixture of 9 P. aeruginosa strains, contained up to 47 antigenic components, many of them being common to the strains of different O-serogroups (immunotypes).