Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.     

Serogrouping of Halophilic Bdellovibrios from Chesapeake Bay and Environs by Immunodiffusion and Immunoelectrophoresis

Little has been reported on the serological relationship of halophilic bdellovibrios (Bd). Immunodiffusion analysis performed with rabbit or mouse Bd antisera developed against eight halophilic Bd isolates and one terrestrial Bd isolate, when reacted with soluble antigen preparations of 45 isolates of halophilic Bd, allowed separation into seven serogroups, which were distinct from the terrestrial isolate. Soluble antigen preparations of prey bacteria, Vibrio parahaemolyticus P-5 (P-5) and Escherichia coli ML 35 (ML 35), exhibited no reactivity with the antisera by immunodiffusion. Immunoelectrophoresis revealed the presence of three distinct antigens in homologous reactions and one shared antigen in heterologous Bd reactions. Shared antigens were noted between halophilic and terrestrial Bd, in addition to between halophilic Bd strains, indicating the possible existence of an antigen(s) which may be shared among all Bd. Again, no shared antigen was noted when P-5 or ML 35 was allowed by immunoelectrophoresis to react with the antisera. Prey susceptibility testing of the seven distinct groups of halophilic Bd, using 20 test prey, produced essentially identical spectra for each group, indicating that this was not a useful technique in delineating the Bd. While immunoelectrophoresis was able to demonstrate an antigen common to all Bd tested, immunodiffusion was able to delineate strains on the basis of a “serogroup specific” antigen. This suggests that immunological tools may serve as important means to study the taxonomy of halophilic Bd, as well as in the formation of a clearer taxonomic picture of the genus Bdellovibrio.     

Antigenic Analysis of Rhizobium japonicum by Immunodiffusion

Immunodiffusion reactions were studied with seven strains of Rhizobium japonicum and three strains of the cowpea miscellany by using antisera against eight of the strains. Most strains yielded only weak precipitin bands when untreated cell suspensions were used as antigens in the diffusions. Ultrasonic disruption or heat treatment of the cells led to stronger bands, and immersion in boiling water for 20 min was used as the standard procedure for preparing these bacteria for immunodiffusion analysis. Heat-labile antigens were detected in only a few strains; the major antigens of all of the strains appeared to be heat-stable. Many of the strains cross-reacted, sometimes in a nonreciprocal manner; unheated cell suspensions cross-reacted more widely but more weakly than the heated suspensions. Heat-treated crushed nodule preparations reacted well in immunodiffusions. The antigens of cultured cell and nodule extract (bacteroid) forms of three strains were compared. In one of these strains, an antigen present in the cultured cells was absent from the bacteroids. Unknown strains present in soybean root nodules were readily identified by immunodiffusion.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V. Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.     

Trypanosoma brucei: Antigenic analysis of bloodstream,vector, and culture stages by the quantitative fluorescent antibody methods

Quantitative direct fluorescent antibody methods were used in antigenic analysis of developmental stages of Trypanosoma brucei brucei strains, most of them having the same variant antigen B, which were derived from a cyclically transmissible stabilate. Antigen-B trypanosomes were used for initiation of cultures in modified Tobie's (Tm) medium and in Glossina morsitans morsitans organ cultures, and for the infective feed of G. m. morsitans. Antisera against antigen-B bloodstream forms and against Tm-grown culture forms were developed in rabbits by inoculations of disrupted organisms mixed (1:1) with complete Freund's adjuvant. The globulin fractions of the antisera were conjugated with fluorescein isothiocyanate, and processed on Sephadex G-25 and DEAE-cellulose columns. The DEAE fractions with 2.0 and 4.7 or 4.8 molar fluorescein:protein ratios were pooled and concentrated twofold.Examination of 109 flies at 30 or 31 days after the infective feed revealed about 18.3% midgut, about 10.1% proventricular, and about 3.7% salivary-gland infections. A salivary gland suspension from one of the infected flies gave rise to a parasitemia in a mouse, and trypanosomes from the first parasitemia were transferred by two 3-day syringe passages into another mouse. Smears were prepared of trypanosomes (antigens B-164, B-167) from the first parasitemias from these two mice, of intact B-antigen trypanosomes, of culture forms (CT) from Tm medium, and of procyclics (CG) from Glossina cultures as well as of midgut (GM), proventricular (GP), and salivary-gland (GS) forms from tsetse flies. All these forms were fixed by one or more of the three following methods: complete fixation (CoFix) by the formalin-NH₄OH-Tween 80 procedure; fixation before affixation to slides (F+); fixation after affixation to slides (F?). The best results with regard to fluorescence intensity and specificity were obtained by using the CoFix technique.Statistical analyses of the fluorescence means of the antigens subjected to direct and inhibition staining gave the following results: (1) CT, CG, GM, and GP forms were antigenically the same. (2) GM and GP trypanosomes from different flies were antigenically indistinguishable. (3) The surface antigen of the variant-B bloodstream trypanosomes was different from these antigens of culture, midgut, and proventricular forms. It differed also from those of metacyclics from two flies and of B-164 and B-167 bloodstream forms. (4) No antigenic differences were found, in preparations fixed by the F? method, between B-164 and B-167 bloodstream trypanosomes and the metacyclics from two flies, one of which served as the source of the salivary-gland trypomastigotes (GS-98) that gave rise to these two bloodstream form antigens. (5) Closer antigenic relationships were noted between B forms and B-164 and B-167 trypanosomes than between B and CT organisms in smears fixed by the F+ technique, but no such differences were discernible in preparations fixed by the F? procedure.     

The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

Schistosoma mansoni: Characterization of antigens in excretions and secretions

Excretory and secretory antigens of Schistosoma mansoni were obtained by in vitro cultivation of worms in Medium H-199, under sterile conditions at 37 C, in the dark, in an atmosphere of 92% air and 8% CO₂. This procedure yielded about 1 μg soluble excretion-secretion products per worm per 24 hr. The composition of the “excretory and secretory antigen” (ESA) preparation is complex. Analysis by isoelectric focusing revealed the presence of about 10 major and about 30 minor protein components. Immunological analysis of the ESA preparation was performed by immunoelectrophoresis. At least five precipitin arcs were seen with infected mouse serum, and seven with rabbit anti-ESA serum. Immunoelectrophoresis of molecular-weight fractions of ESA showed a total of 17 different antigens. One of these antigens was excreted exclusively by female worms. The antibody response in rabbits to preparations obtained by homogenization of adult worms, or by extraction of the tegument, was very different from the response to excretory and secretory antigens. Considerable cross-reactivity between these preparations did, however, occur.     

Thermostability of Antigens Associated with Serotype of Rhizobium japonicum

The antigens associated with serologically distinct strains of Rhizobium japonicum were found to differ in heat sensitivity. Cell preparations from 4 out of 12 strains retained agglutinability, and 1 out of 5 retained antigenicity after they were heated to 120 C. Antigenicity was reduced in most strains after heating to 100 C for 30 min, but agglutinability was not affected by this treatment. This suggests that the antigens are protein-polysaccharide-lipid complexes described for O-type antigens. Cells of strain 46, however, retained both agglutinability and antigenicity after heating to 120 C for 1 hr, and thus a protein in its structure seems improbable. Antigens associated with bacteria from soybean nodules responded to heat treatment in essentially the same manner as those from the same strain grown in nutrient broth. Certain serotypes showed a tendency to agglutinate spontaneously. A heat treatment of 100 C for 30 min, to remove nodule debris and destroy certain blocking antigens, did not interfere with the agglutination reaction. Live cells induced antiserum in rabbit to a higher titer than did heat-treated cells.     

Mitogenic and antigenic activity of Plasmodium falciparum in primate and rodent lymphocytes

Goldenser J., Marva E., Spira D. T., Gabrielsen A. A. and Jensen J. B. 1985. Mitogenic and antigenic activity of Plasmodium falciparum in primate and rodent lymphocytes. International Journal for Parasitology15: 435–440. Considerable reaction of human leucocytes to a wide range of concentrations of plasmodial preparations derived from in vitro cultures of Plasmodium falciparum was observed. Highest responses were recorded after 6 days in culture. This differed from the response to PHA or CON-A which peak with a narrow range of concentrations after 3 days in culture. Parasitized erythrocytes (PE) or parasites released from PE as well as soluble antigens obtained from the particulate preparations had a pronounced mitogenic activity which was unaffected by heating to 56°C for 1 h. Peripheral lymphocytes from man and monkey but not from rats reacted to P. falciparum preparations. Spleen cells obtained from normal rats did not react towards any P. falciparum preparation. Spleen cells of rats immune to P. berghei, responded to normal human erythrocytes but the response against P. falciparum antigens was much higher, indicating cross-reactivity with genus specific antigens. The combination of experimental procedures using human peripheral and rat spleen lymphocytes is suggested for differentiation between mitogenic and antigenic activity. Heat inactivation of some proteases present in the plasmodial preparations, while retaining mitogenic activity, may enable further purification of the mitogenic factors.     

Immunization of mice against infection with Taenia taeniaeformis using various antigens prepared from eggs,oncospheres, developing larvae and strobilocerci

Rajasekariah G. R., Rickard M. D. and Mitchell G. F. 1980. Immunization of mice against infection with Taenia taeniaeformis using various antigens prepared from eggs, oncospheres, developing larvae and strobilocerci. International Journal for Parasitology10: 315–324. Antigens were collected during in vitro incubation of oncospheres, 3-week-old larvae and strobilocerci of T. taeniaeformis. Supernatants of these in vitro products centrifuged at 500 g contained antigens which stimulated a significant degree of protective immunity when injected into mice. However, centrifugation of the strobilocercus preparation at 4500 g yielded supernatants which failed to induce immunity. Suspensions of eggs and oncospheres disrupted by sonication stimulated a high level of immunity as also did 4500 g supernatants of the sonicated preparations. Centrifugation of sonicated oncospheres at 100,000 g yielded a supernatant which stimulated significantly less immunity than the 4500 g supernatant, although the protective capacity was not totally abolished. The pellet from 100,000 g centrifugation of sonicated oncospheres induced almost absolute immunity. These results are consistent with the suggestion that the ‘functional’ antigens in the preparations tested may initially be membrane-associated or particulate in nature and that sonication causes partial solubilization. Supernatants prepared from homogenised strobilocerci and centrifuged at 4500 g also stimulated protective immunity and presumably contain soluble antigens. No evidence is available to suggest whether or not the strobilocercus antigens which stimulated protective immunity are identical to those found in oncospheral preparations. Immunity was stimulated by subcutaneous, intraperitoneal and intramuscular injections of antigen and both Freund's complete adjuvant and Bordetella pertusiss vaccine were effective as adjuvants. Using sonicated oncospheres, a high level of immunity was stimulated without the use of adjuvant.     

Effectiveness of Different Frankia Cell Types as Inocula for the Actinorhizal Plant Casuarina

The soil bacterium Frankia of the Actinomycetales, capable of forming N₂-fixing symbiotic root nodules on a diverse array of actinorhizal plants, has several morphological forms when grown in pure culture. Fresh hydrated preparations of whole cells, hyphae, and spores were all infective on seedlings of Casuarina at different dilutions. Desiccated hyphae showed no infection capacity, while desiccated spores remained infective, although at a reduced level. On the basis of most-probable-number statistics, spore suspensions were 3 orders of magnitude more infective than hyphae.     

Development of Stable Vibrio cholerae O1 Hikojima Type Vaccine Strains Co–Expressing the Inaba and Ogawa Lipopolysaccharide Antigens

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co–express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba– and Ogawa–LPS antigens which are preserved after formalin–inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin–inactivated whole–cell vaccine preparations of these strains elicited strong intestinal IgA anti–LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole–cell vaccines.     

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: Interspecific and intraspecific differences in infectivity for insects

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.     

Failure to vaccinate lambs against Haemonchus contortus with functional metabolic antigens identified by immunoelectrophoresis.

Metabolic products from Haemonchus contortus larvae cultured in vitro from the infective third to fourth stage were collected and concentrated. Chromatographic and immunoelectrophoretic analyses were made to study the numbers and activity of antigens in the metabolic products derived from the in vitro cultured larvae. Three-month-old lambs were given a series of injections of metabolic antigens with and without adjuvant at dose rates of 0·05, 0·5 and 5·0 mg antigen protein per injection. These animals and saline injected controls were each challenged with 3000 H. contortus infective larvae after the last antigen injection and killed 35 days later. No difference was seen in the faecal worm egg counts or the differential worm counts among the vaccinated and control animals. The antigen preparation of worm metabolic products conferred no resistance to challenge infection with the parasite.     

Expression of the primary biliary cirrhosis antigens in yeast: Aspects of mitochondrial control

The mitochondria of 21 yeast strains were tested for the expression of primary biliary cirrhosis (PBC) specific antigens. The amounts of the antigens in the mitochondrial preparations varied with the strains. Genetic analysis of the strain differences in antigen expression indicated nuclear control which was complex. Those strains expressing the least amounts of antigens exhibited coagulating mitochondria in organellar preparations. Additional evidence relating expression of antigens to the physiological/structural state of mitochondria was that cells grown in the presence of the mitochondrial uncoupling agent, 2,4-dinitrophenol (DNP), failed to produce any antigens, and that glucose repression of mitochondria suppressed antigen expression. Blockage of mitochondrial protein synthesis either throughpetite mutation or by culture in the presence of erythromycin decreased the content of antigens in the mitochondria but did not competely block antigen production. The presence of the PBC antigen in the mitochondria of these cells with nonfunctional mitochondrial synthesizing machinery further indicates that these antigens are cytoplasmically synthesized. Analysis of the pre- and postmitochondrial fractions of all homogenates confirmed that the antigens are not only cytoplasmically synthesized but also have an extramitochondrial location in cells, probably in the plasma membrane.     

Chemical analysis of exocellular, acid polysaccharides from seven Rhizobium strains

A comparative, chemical analysis of the acid exopolysaccharides from seven Rhizobium strains, involving the taxonomic groups Rhizobium meliloti, Rh. trifolii, Rh. phaseoli, and Rh. leguminosarum, is presented. Apart from the polysaccharide from Rh. meliloti, which is known to lack uronic acid, no significant differences in the carbohydrate composition were found. The two non-nitrogen-fixing strains [infective (Coryn), and non-infective (Bart A)] gave polysaccharides which differ from those produced by the infective and nitrogen-fixing strains in the detailed structural features. This difference is expressed in the pattern of periodate oxidation and cation-binding capacity.     

Potency of adenovirus vaccines: comparison of unconcentrated, concentrated, and soluble antigen preparations

Five adenoviruses were effectively concentrated and partially purified by methanol precipitation. The soluble antigens from one adenovirus concentrate were obtained by adsorption to and elution from calcium phosphate. Formaldehyde-inactivated vaccines prepared from the virus concentrates were more antigenic in guinea pigs than a National Institutes of Health reference vaccine or one prepared from unconcentrated adenovirus. The soluble antigens also proved to be antigenic. The data indicate that adenovirus vaccines of superior potency can be prepared from concentrated virus preparations and that the extracted soluble antigens are immunogenic.     

Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa

Chitosan and its derivative water soluble Chitosan oligosaccharide are used in a variety of applications in pharmaceutical preparations. In this study, 2 wild (ATCC 15729 and PAO1) and 2 mutant strains (PT121 and PT149) of P. aeruginosa are investigated for drug-drug interactions in vitro. 10 antimicrobial agents (antibiotics) are combined with different degree of deacetylated Chitosans and Chitosan oligosaccharide. All the chitosans show synergistic activity with sulfamethoxazole, a sulfonamide antimicrobial agent. It is interesting to observe that the MIC value for the MexEF-OprN overexpressing mutant strain of P. aeruginosa is 5 fold higher than the other strains under investigation suggesting a possible role of this efflux pump in Sulfamethoxazole efflux. The findings suggest on the use of chitosans as enhancing agent in combination with antibiotics in pharmaceutical preparations.     

Presence of soluble SLA histocompatibility antigen in pig plasma.

The major histocompatibility antigens of the pigs (SLA 1 and SLA 15) were solubilized by papain and then iodinated according to Greenwood's chloramine T method. These antigen preparations were used in radioimmunoassays for the detection of soluble inhibitors in pig plasma. Specific soluble substances were demonstrated in addition to a certain amount of cross-reactivity with other so far unidentified antigens.     

Hydrolytic Enzyme Production by Rhizobium

Cellulase and hemicellulase activity was detected in temperate (infective and noninfective) and tropical strains (infective) of Rhizobium. Hydrolytic enzymes were initially detected by a cup-plate assay. The presence of cellulase and hemicellulase was confirmed by viscometric assay. Implications of the presence of these enzymes in Rhizobium are discussed.