Heat-stable Antigen Research Articles

Properties of heat-stable antigen in the culture supernate of Haemophilus paragallinarum.

To analyze the antigcnic component of Haettnojohilus paragallinarutn (Hpg), serological and immunological properties of a heat-stable antigen in the culture superrtateof Hpg were studied. At least two different antigens were found in the culture supernateof various Hpg strains by the agar get precipitation (AGP) test. They NVCT( separated ir?tc>two fractions by get filtration. -[lte Iteat-stable antigen was eluted at void volume by getfiltration with Sepharose 6B and 2B, but not any other. The heat-stable antigen did notlose antigenicity when it was lacated at l2lC for 30 minutes. The AGP test revealed tltatthe heat-stable antigen was not type-specific to az?y Hpg serotype. Moreover, lipopolysaccharide (LPS), which had I>een isolated from Hpg cells by XVestphals methods, wasfound to have antigenicity common to Hpg serotypes. In the AGP test, tlae t?eat-stableantigen showed an identity reaction to the LPS product. Neither heat-stable antigett inthe culture supernate nor LPS arntigen of Hpg strain 221 presented a protective effect wlneninjected into chickens, or agglutinated chicken red blood cells. Furthermore, no measurable hemagglutination-in1?ibition antibody was found in chickens immunized w"itlt botltantigens. These results suggested that heat-stable antigen in the culture supernate ofIlpg might be composed of LPS.

Serological Diagnosis of Bacterial Kidney Disease of Salmonid (BKD): Immunodiffusion Test by Heat Stable Antigen Extracted from Infected Kidney

An immunodiffusion test with heat stable antigen extracted from the kidney of infected fish is described as a method of diagnosis of BKD.Specific precipitin bands were formed within 24 hours when the rabbit anti-serum reacted with the extract of affected parts of the kidney containing heat stable specific BKD antigen.No precipitin bands were formed with heat extract of the kidney of healthy fish and heat extract of cells of other fish pathogenic bacteria, i. e. Aeromonas salmonicida, Vibrio anguillarum, etc. From the above findings, it was suggested that the correct diagnosis of BKD could be easily made in distant diagnostic laboratory by the sending of heat treated field specimens.

Immunological procedure for the rapid and specific identification of Coccidioides immitis cultures.

An immunological procedure for the rapid and specific identification of Coccidioides immitis isolates has been developed. The specificity of the procedure is based on the fact that C. immitis produces antigens that are not produced by morphologically similar fungi. The procedure involved the transfer of heavy mold-form inocula to flasks that contained small volumes of brain heart infusion broth. Shake cultures were grown at 25 degrees C on a gyratory shaker at 150 rpm. The concentrated supernatant antigens so obtained from broth cultures were tested in parallel with coccidioidin by a microimmunodiffusion technique against C. immitis antiserum. The ability of the immunological procedure to identify C. immitis cultures was evaluated by testing the supernatant antigens of 166 unknown isolates, many of which, by gross or microscopic examination or both, resembled C. immitis or belonged to the family Gymnoascaceae. Each culture was also identified by conventional laboratory procedures. Comparative evaluation showed that the immunological test for C. immitis was 100% sensitive and specific. The diagnostic precipitin bands that were produced by the interaction of the C. immitis supernatants and antisera were shown to consist of (i) a heat-stable antigen comparable to that active in the tube precipitin test and (ii) two heat-labile antigens, one of which was associated with complement fixation reactivity. With pure cultures, this immunological procedure permitted the identification of C. immitis isolates within 5 days.

Antigens of Haemophilus influenzae.

Three antigenic preparations were obtained from a non-capsulated strain of Haemophilus influenzae by ultrasonic disintegration, hot phenol extraction and from a fluid culture. They were designated H. influenzae cytoplasmic antigen (H(1-5); H. influenzae cell wall antigen (HCW); and H. influenzae culture filtrate antigen (HCF). Studies showed that H(1-5) antigen contained heat stable and heat labile components. The heat stable fraction stained positively for polysaccharide, had a positive limulus lysate test and there was immunological cross-reactivity between this and heat stable fractions of HCW and HCF. Limulus lysate assay indicated the presence of endotoxin in HCW and HCF preparations. Heat stable as well as heat labile antigens of H. influenzae should be given consideration in future studies regarding the pathogenicity of this organism in the lower respiratory tree. The specificity of the heat stable antigen of H. influenzae needs to be determined.

Antibody-mediated immobilization of Campylobacter fetus: inhibition by a somatic antigen.

Immobilization tests were conducted on a wild-type strain of Campylobacter fetus subsp. intestinalis and on a mutant lacking an antiphagocytic cell surface component. Highly effective immobilization of the mutant, both as single cells and clumps of cells, was produced with an antiserum containing antibodies specific for the flagellar hook and filament and for the O antigen. Damage to flagellar hooks after reaction with this antiserum was observed only with cells of the mutant. Single-cell immobilization of the mutant was also produced with an antiserum specific for a heat-stable somatic antigen which was distinct from the O antigen and was exposed on the cell surface only of the mutant. Minimal immobilization of the wild strain was brought about by either of these antisera. It was shown also that O antibodies had no effect on the motility of either the wild strain or mutant. These findings indicate that antibody-mediated immobilization may be brought about by effects on the flagellar hook or cell body, as well as on the flagellar filament. Furthermore, the protection from immobilization afforded the bacterium by the antiphagocytic surface structure suggests a dual function for this virulence factor in the infected animal.

Immunoelectrophoretic analysis of Mycoplasma mycoides var. mycoides.

Acrylamide gel electrophoresis was used to show the similarities and differences in the membrane proteins of two vaccine and two virulent strains of Mycoplasma mycoides var. mycoides. Immunoelectrophoretic (IEP) analysis was also used to partially characterize the associated antigens. Antibody spectra to the antigens of M. mycoides differ in rabbit, pig, and cattle sera. Rabbits produce better precipitating antibody against the anodic migrating protein mycoplasma antigens than cattle and pigs as seen in IEP. However, rabbit anti-M. mycoides serum did not show precipitating antibody against the heat-stable carbohydrate antigen. As judged by IEP, the major carbohydrate antigen extracted from the media, or boiled whole organism, is similar to that present in the sera-infected cattle and knee joints of calves. This carbohydrate antigen has a cathodic migration in IEP at pH 8.6. Periodate oxidation, classically used to destroy carbohydrate, also destroys most of the protein antigens. Heating the antigens to 56 C for 10 min destroys many of the noncarbohydrate antigens and 100 C eliminates all but the carbohydrate antigen. Extraction of M. mycoides with chloroform-methanol, phenol, ethanol, or ethanol-acetone reduced or eliminated most of the protein antigens. Some of the isolated antigenic fractions of M. mycoides were tested to determine their activity in the diagnostic complement fixation test for contagious bovine pleuropneumonia and their inhibitory effect in this test by using bovine anti-M. mycoides antisera having precipitating antibody and circulating antigen. The complement fixation antigen is not the galactan, cannot be extracted by chloroform-methanol, but is stable to boiling at 100 C and may be extracted by phenol and partially precipitated by ethanol-acetone.

Specificity of bacteriophages and antiserum forPseudomonas pisi

Abstract Of 47 isolates from peas including reference strains and cultures suspected of being Pseudomonas pisi, 36 were considered to be typical of that species, on the basis of phage-, sero-, and symptom-type. Eight were similar to Ps. syringae and three were non-pathogenic Pseudomonas spp. The specificity of phage and serological techniques was further tested on a collection of 81 isolates from 27 Pseudomonas species. Phage El (NZ) was highly specific to Ps. pisi. Phage Pg26 (Canadian) cross reacted to a limited extent (6.5% of isolates tested). Antiserum tests for the heat-labile antigen showed 26% cross reactions and for the heat-stable antigen 12% cross reactions. On the basis of serological tests alone (tests for the heat-stable antigen), isolates from three species were indistinguishable from Ps. pisi. On the basis of combined phage and serology tests only one species would have been confused with Ps. pisi. For the routine identification of Ps. pisi, serological tests for the heat-stable antigen an...

Fowl Cholera: Gel Diffusion Precipitin Test for Serotyping Pasteurella multocida from Avian Species

SUMMARY Heat-stable antigens extracted with formalinized saline were used in the gel diffusion precipitin test to group Pasteurella multocida associated with fowl cholera into 5 serotypes. Of 258 field isolants tested, 33 were type 1, 157 were type 3, 28 were type 4, 4 were type 5, and 4 were type 6; 27 isolants reacted with type 3 antiserum and to a lesser extent with type 4 antiserum, and 5 isolants did not react with any antisera. Bacterins prepared with P. multocida serotypes 1, 3, and 4 induced good immunity in chickens and turkeys against organisms of the same serotype. A bacterin prepared with serotype 4 culture induced as good immunity against a serotype 5 challenge-exposure culture as was induced with the homologous bacterin. The serological behavior of the heat-stable antigens was compared by gel diffusion and immunoelectrophoresis with partially purified lipopolysaccharide-protein complex (endotoxin) that had induced specific immunity in turkeys. Lines of identity were observed in the gel diffusion test, but a slight difference was observed with immunoelectrophoresis. The heat-stable antigen was demonstrated in the serum of a turkey with acute fowl cholera and in the blood of turkeys that died of the acute disease.

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.

Studies on nondefective adenovirus-simian virus 40 hybrid viruses. I. A newly characterized simian virus 40 antigen induced by the Ad2+ND 1 virus.

The nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), does not induce heat-labile SV40 T antigen but does induce a previously uncharacterized heat-stable SV40 antigen-the SV40 "U" antigen. This antigen is detectable by both immunofluorescence and complement fixation by using sera from hamsters with SV40 tumors. Sera from hamsters bearing SV40 tumors can be divided into two groups, those that react with both SV40 T and U antigens (T(+)U(+) sera) and those that react with SV40 T antigen only (T(+)U(-) sera). SV40 U-specific sera from monkeys immunized with Ad2(+)ND(1)-infected cells do not react with SV40 T antigen by immunofluorescence but do react with an antigen in the nucleus of SV40-transformed cells and with an early, cytosine arabinoside-resistant antigen present in the nucleus of SV40-infected cells. A heat-stable SV40 antigen detectable by complement fixation with T(+)U(+) hamster sera is present in extracts of SV40-induced hamster tumors and in cell packs of SV40-infected or -transformed cells. SV40 U-antigen synthesis by Ad2(+)ND(1) virus is partially sensitive to inhibitors of deoxyribonucleic acid synthesis, whereas U-antigen synthesis by SV40 virus is an early cytosine arabinoside-resistant event. As an early SV40 antigen differing from SV40 T antigen, U antigen may play a role in malignant transformation mediated by SV40.

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.

Further studies on the phage sensitivity and the determination of phytopathogenic Pseudomonas spp.

Summary.An interim determinative scheme for phytopathogenic Pseudomonas spp. by a variety of cultural and biochemical tests was suggested by Lelliott, Billing & Hayward (1966) following the study of 156 green fluorescent phytopathogenic pseudomonads. The phage sensitivity of these and 80 additional cultures to 20 phages from a variety of sources are described. Information on the morphology of these phages is now available (Bradley, 1963a). A few nutritional tests which seem to be of diagnostic value at the species level are also included and a relationship between the presence of a heat stable antigen (Lovrekovich et al., 1963) and phage sensitivity patterns is discussed.

ジフテリア菌ならびにその他のコリネバクテサウムの血清学的分類とジフテリアトキソイドによるアレルギー反応に関する研究

The antigenic structures of Corynebacterium including C. diphtheriae and others were studied by agglutination reaction and it was confirmed that the strains so far tested contained heat-labile and Oinagglutinable antigen and heatstable antigen. The heat-labile antigen was rather type-specific and strainspecific and heat-stable antigen was cross-reacting to almost all of the tested strains including C. belfanti, C. hofmannii, C. xerosis, and C. ulcerans.In this connection the experimental allergic reaction of guinea pig was investigated and it was confirmed that heat-stable antigen other than toxoid could be responsible for the allergic reaction to the crude toxoid in the market, but it did not sensitize the guinea pig against the purified diphtheria toxoid in the purity of 3300 Lf per mg. P. N.And it was confirmed that the guinea pig could be sensitized against the diphtheria toxoid in the purity of 3300 Lf per mg. P. N.

Immunofluorescence in the study of Marek's disease. I. Detection of antigen in cell culture and an antigenic comparison of eight isolates.

The indirect fluorescent-antibody (FA) test was applied to the detection of Marek's disease (MD) antigen in cell culture and antibody in the serum of birds. For the detection of antigen, sera were obtained from birds hyperimmunized with the JM strain of MD. MD antigen could be detected in the nucleus and in the cytoplasm of duck and chick embryo fibroblasts and in those of chick kidney cells infected with material known to contain the MD virus. Uninoculated cultures of chicken cells were always free of MD antigen. When chick kidney cells were infected with a stock cellular preparation of MD virus, infected cells could be detected after 24 hr with the FA test. At this time no cytopathological areas were seen by conventional light microscopy. By 7 days after infection, the same number of infected areas were detected by both methods, and the fluorescent areas coincided with the cytopathological areas. This indicates that the fluorescent areas and the areas with cytopathology are caused by the same agent. A straight-line relationship between the dilution of inoculum and the number of fluorescent or morphological foci obtained indicates that one infectious unit produced one fluorescent or morphological focus. In addition, this time sequence study confirmed the cell association of the virus and demonstrated the cell-to-cell spread of infection. Cell cultures inoculated with eight different isolates of MD were tested in all combinations with sera prepared against the same isolates. The antigens were indistinguishable from one another, indicating that either the strains are antigenically identical or there is a common antigen or contaminant in all of them so that they stained equally well. The FA test can detect MD antigen before cytopathological areas develop in cell culture; however, the small size of the area usually examined precludes its use in initial isolations in which only a small number of infectious units are present in the inoculum. MD-infected cells contain a heat-stable antigen similar to that found in herpes simplex-infected cells.

Immunological studies in ulcerative colitis. IV. Origin of autoantibodies.

The incidence and height of antibody titers to colon, assayed by indirect hemagglutination with a heat stable colon extract from germ free rats, is significantly higher in sera from patients with ulcerative colitis than in those from healthy controls or from patients with amebic liver abscess or dysentery. While sera from ulcerative colitis patients and controls are indistinguishable in regard to incidence and height of antibody titers to Forsman antigen, Staphylococcus aureus S 209, Clostridium difficile, and several common strains of E. coli, they have elevated titers and increased incidence of antibodies to a heat stable antigen of E. coli O14. Patients with amebic dysentery have normal titers of such antibodies. Absorption of patients' sera with E. coli O14 antigen inhibits the colon directed hemagglutination reaction in approximately 30% of the cases tested. Likewise, the anti-E. coli O14 reaction can sometimes be inhibited with the colon extract. Other E. coli strains and other bacteria are inactive or have only weak inhibitory activity. Hemagglutination inhibition experiments show that germ free rat colon and E. coli O14 contain common structures, depicted by antibodies in the patients' sera. This pattern of reactivity closely resembles that seen in rats made autoimmune to colon by injection of newborn rabbit colon. E. coli O14 is known to carry a heterogenetic antigen present in lower concentration (or activity) in most Enterobacteriaceae. Hemagglutination inhibition experiments with rabbit antisera to E. coli O14 suggest that the antigen common for E. coli O14 and colon is related to this heterogenetic antigen. The findings imply that this antigen, which is constantly present in low concentrations in the human colon, may give rise to anticolon antibody formation in ulcerative colitis through breakage of tolerance. Since this antigen is present in healthy individuals as well, additional factors are required to explain the induction of anti-colon autoimmunity in ulcerative colitis.

Characterizion of Mycoplasma pulmonis variants isolated from rabbits. II. Basis for differentiation of antigenic subtypes.

The antigen composition of Mycoplasma pulmonis variants was studied by complement-fixation, agar-gel diffusion, and growth-inhibition tests. Two classes of complement-fixing antigens were demonstrated for M. pulmonis strains 47 and 63: (i) cross-related, heat-labile, water-soluble antigens, and (ii) high-titered, subtype-specific, heat-stable, water-soluble antigens. Lipid antigens prepared by organic solvent fractionation were low-titered antigens and showed little specificity. With the aid of agar-gel double-diffusion plates, the subtype-specific antigens were found to be precipitated by trichloroacetic acid and to be stable to periodate, but they were inactivated by pronase. Pronase-stable, periodate-labile precipitating antigens were observed as common components between the two variants. Antisera prepared with boiled antigens were found to be serologically active on gel diffusion but lacked neutralizing ability in growth-inhibition tests. Each of three strains of M. pulmonis (47, 63, ATCC 14267) could be identified as a variant because each strain possessed immunologically distinct heat-stable subtype-specific antigen(s).

Some properties of the complement-fixing antigens of the agents of trachoma and inclusion blennorrhoea and the relationship of the antigens to the developmental cycle.

SUMMARY: The viruses of trachoma and inclusion blennorrhoea, like other members of the psittacosis-lymphogranuloma group, contain heat-stable and heat-labile complement-fixing antigens. The heat-stable antigen, which is soluble in ether and partly destroyed by periodate, appears to be chemically similar, as well as serologically related to, the lipopolysaccharide-protein complex shared by other viruses of the group. Complement-fixing group antigen was detected at all times during one cycle of multiplication of a strain of inclusion blennorrohea virus in HeLa cells. The amount of cell-associated antigen, about half of which sedimented under the same conditions as mature elementary bodies, remained constant for the first 16 hr. after adsorption; after 16 hr. it increased linearly, reaching a maximum 28 hr. after adsorption. The group antigen probably forms at a stage in the developmental cycle when large forms predominate and is later distributed among the smaller infectious elementary bodies.

Studies on Trachoma

SummaryThis paper reports the isolation of strains of elementary bodies from trachoma cases in Saudi Arabia and Egypt by inoculation of the yolk sacs of chick embryos. Nineteen strains were obtained in 45 attempts using conjunctival scrapings from children who were shown to have typical inclusions at the time of the scraping. Nine of the strains were subjected to a series of tests which showed that they were indistinguishable morphologically from the elementary bodies of the psittacosis-lymphogranuloma group of viruses, that they were not sensitive to streptomycin, that they were free of other viruses and of pleuropneumonia-like organisms, that they were toxic for white mice by the intravenous route, and that they contained the heat stable antigen which is common to the psittacosislymphogranuloma group of viruses. Although each strain was capable of multiplication in yolk sac membranes of chick embryos, irregularities of growth were frequently observed, being more characteristic of certain strains, than others.Viability of the trachoma elementary bodies in conjunctival scrapings persisted at -60°C for many months if the specimens were stored in a medium containing sucrose, glutamate, and phosphate. One of the strains induced conjunctivitis in a chimpanzee whose conjunctival cells showed typical inclusions at the height of the conjunctival reaction. A laboratory worker contracted severe trachoma probably as a consequence of air-borne droplets arising from manipulations of concentrated suspensions of the elementary bodies.

Studies on immunity to Asiatic cholera. X. Heat stable cell wall and intracellular antigens of Vibrio cholerae independent of agglutinogens and demonstrable by passive hemagglutination.

not follow that such superficially located antigens reflect completely the antigenic mosaic of the entire cell. If not, then antigens other than agglutinogens may be present in the cell, and an antigenic structure concerned only with agglutinogens is incomplete. In studies on the mouse protection test for antibody protective against challenge inoculation with Vibrio cholerae (Burrows, Mather, Elliott and Havens, 1947), it was found that an appreciable amount of mouse protective antibody, one-fourth to one-third of the initial titer, was demonstrable by the passive protection test in antiserums homologous to the challenge vibrio strain but exhausted of homologous agglutinins. It was concluded that Protective antibody was associated with antibody to heat stable vibrio antigen, but was not necessarily identical with O agglutinin. Similar results were reported later (Burrows, 1953) when it was shown that an analogous partial protection against enteric infection of the guinea pig with V. cholerae could be obtained by active immunization with O vaccine prepared with a noncholera vibrio strain whose

Immunologically reactive substance from Schistosoma mansoni

A method is described for the preparation of a nondialyzable aqueous extract of adult Schistosoma mansoni worms which were experimentally propagated in mice. The cell-free extract of the schistosomes is chemically complex and qualitative tests have established the presence of carbohydrate and nucleoprotein components. It represents about 10 to 20 % of the weight of the worms and contains 7 to 9 % organically-bound nitrogen by weight. It contains a relatively heat stable antigen which is reactive in the complement-fixation test with serum of human S. mansoni disease, but its value in diagnosis of human schistosomiasis has not been investigated. The preparation reacts in the complement-fixation test with a heterophile substance present in the serum of the normal rabbit. The significance of the latter reaction is not understood. Serums of normal mouse, guinea pig, human, human serums positive serologically for syphilis and of cattle positive for leptospirosis do not contain similar heterophile-like substances.