Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

Utilization of cathodically-produced hydrogen from mild steel byDesulfovibrio species with different types of hydrogenases

Summary Desulfovibrio (D.) vulgaris Hildenborough with a highly active Fe-containing periplasmic hydrogenase,D. salexigens British Guiana with a Fe–Ni–Se periplasmic hydrogenase, andD. multispirans with a Fe–Ni cytoplasmic hydrogenase utilized cathodically-produced hydrogen from mild steel as the only energy source for activity and growth. Changes on the mild steel surface occurred during growth of these bacteria. The concentration of iron sulfide, a corrosion product of mild steel, increased over time, andDesulfovibrio species had an active hydrogenase when they were grown in lactate/sulfate media. This hydrogenase may be any of the three types found in the genus,Desulfovibrio. The concentration of iron in the media affected the production and activity of the Fe-hydrogenase fromD. vulgaris Hildenborough. With an iron-limited medium, the specific activity and the total amount of the periplasmic hydrogenase was less than found with a non-iron limited media.     

Serological Relationship of Meloidogyne incognita and M. arenaria

Eight to ten precipitin bands were formed in a double immunodiffusion system comparing antigens of adult females of Meloidogyne incognita and M. arenaria. Most of the precipitin bands, based on band position and coalescence, were common to both species. Antiserum specific for M. incognita was prepared by cross absorption. Two populations of M. incognita were serologically identical, whereas two populations of M. arenaria differed slightly with respect to one weak precipitin band.     

CHARACTERIZATION OF POLLEN ANTIGENS FROM AMBROSIA L. (COMPOSITAE) AND RELATED TAXA BY IMMUNOELECTROPHORESIS AND RADIAL IMMUNODIFFUSION

The pollen antigens of various Ambrosia and related species were studied to learn whether substances closely related to antigen E (the major allergen of Ambrosia artemisiifolia) were present. After conventional immunoelectrophoresis, pollen extracts from six Ambrosia species each produced at least one pronounced precipitin line with antiserum for purified antigen E. Electrophoretic mobility was the same for several species (A. artemisiifolia, A. bidentata, A. psilostachya, and A. trifida) but was relatively lower for A. acanthicarpa and A. ambrosioides. Precipitin rings were also produced when pollen extracts of the various Ambrosia species were subjected to radial immunodiffusion in agarose which contained antiserum for purified antigen E. There was great variation among the Ambrosia species with respect to precipitin ring diameters. The variation may be due to differences among species in content of the antigen E-like substances or to altered interaction with the immobilized antibody. Crossed (2-dimensional) immunoelectrophoresis was shown to be useful for characterizing Ambrosia pollen antigens. Pollen extracts from A. artemisiifolia produced eight pronounced precipitin bands and at least eight faint, relatively fast-moving bands after crossed immunoelectrophoresis with antiserum against a whole pollen extract from the same species. One of the pronounced bands contained antigen E.     

Biochemical and Immunological Study of Cell Envelope Proteins in Sulfate-Reducing Bacteria

The envelope proteins of 5 strains of the genus Desulfotomaculum and 12 strains of the genus Desulfovibrio were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The Desulfovibrio strains exhibited a typical gram-negative cell envelope, whereas the cell envelope of Desulfotomaculum strains appeared to be gram-positive. A close relationship between strains of Desulfotomaculum nigrificans was observed. A comparison between different species of Desulfotomaculum revealed some degree of similarity between Desulfotomaculum nigrificans and Desulfotomaculum ruminis, whereas Desulfotomaculum orientis seemed unique. The strains of Desulfovibrio salexigens were quite different from the strains of the other species of Desulfovibrio. In two of the strains of Desulfovibrio desulfuricans, a species-specific antigen was observed. The strains of Desulfovibrio vulgaris, Desulfovibrio africanus, and Desulfovibrio gigas and one strain of Desulfovibrio desulfuricans exhibited a similar outer membrane protein profile and also showed very similar antigenic reactions.     

Precipitin studies for the identity of antigens in different species of Sporotrichum

Summary 1) A simple technique for the preparation of dissolved autoclaved antigens and of sonic vibrated antigens, the immunisation of rabbits, and testing methods by precipitation in agar medium and in capillary tubes are described.2) Five different strains ofSporotrichum belonging to three species,S. Schenckii, S. Beurmanni, andS. asteroides are studied as to their precipitin lines formation in agar medium.3) The similarity in precipitin lines formation is interpreted to demonstrate the antigenic identity of these differentSporotrichum species.4) Comparable results with minor differentiation but in major sensitivity are obtained by precipitin tests in capillary tubes.5) Temperatures of 4° C are shown to stimulate intensity of precipitin lines.6) Dissolved sonic vibrated antigens alter the precipitin formation by changing and intensifying the precipitin lines.7) Absorption experiments with dissolved antigens give unsatisfactory results if precipitin tests in capillary tubes are used.This work was done during a Fulbright Fellowship spent with Dr.Norman F. Conant, Department of Bacteriology, Duke University School of Medicine, Durham, N.C.     

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。     

Serological investigations of cell adhesion in the slime molds,Dictyostelium discoideum,Dictyostelium purpureum,and Polysphondylium violaceum

1. Antibodies to slime molds were produced by injecting D. discoideum and D. purpureum amebas from 48 hour cultures into rabbits. 2. Anti-D. discoideum and anti-D. purpureum sera caused agglutination of homologous amebas from 24 to 26 hour cultures, agglutination of certain heterologous amebas from 30 to 36 hour cultures, and agglutination of all heterologous amebas from 43 to 48 hour cultures. 3. The data show that new surface antigens are formed in cultures after 26 hours and it is suggested that the new antigens are concerned with cell adhesion. 4. The probable role of surface antigens in the interaction of cells of different species of slime molds was discussed.     

Immunodiffusion test for diagnosing basidiobolomycosis

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ° C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.     

Serological analysis and pathogenic potentials of Wangiella dermatitidis isolated from bats

Methods for the preparation of antigens from clinically isolated cultures of Aspergillus were standardized. Sera from 25 suspected cases of pulmonary aspergillosis were tested against antigens prepared by us, from 4 strains of A. fumigatus and one strain of A. flavus, using the Ouchterlony double diffusion and immunoelectrophoretic techniques.Of the 25 sera tested, 18 reacted positively with antigens of A. fumigatus, one with A. flavus and 2 with both these species. Antigens of two non-pathogenic Aspergilli included in the study failed to react with any of the sera. Our antigen preparations gave more numerous as well as sharper precipitin lines than the commercial Bencard antigens which were used for comparison. Moreover, mycelial antigens from 48 to 96 h old cultures revealed precipitin lines comparable to that of the routine, 4 week old culture filtrate antigens, thus suggesting that the incubation period for obtaining antigens could be cut down considerably.Memoir No. 323 from the Centre of Advanced Study in Botany.Deceased     

Complementary Methodologies To Identify Specific Agrobacterium Strains

Serological techniques and restriction enzyme cleavage patterns of total DNA were used to differentiate strains of Agrobacterium spp. Forty-five wild-type and plasmid-cured Agrobacterium strains were tested by immunodiffusion and immunofluorescence against polyclonal antisera to a crude ribosome preparation from Agrobacterium strains K84, U11, B6, A323, NT1, and C58. In immunodiffusion gels, these antisera reacted only with water-phenol extracts of the homologous strain, producing a single, strain-specific precipitin line. In contrast, when the same antisera were used in immunofluorescence staining, cross-reactions occurred with a limited number of heterologous Agrobacterium strains. However, the cross-reacting heterologous cells fluoresced generally less brightly than the homologous cells. When the EcoRI-digested DNA profiles from the same Agrobacterium strains were compared, 34 distinct cleavage patterns were observed. The DNA profiles were the same for all strains sharing a common chromosomal background and correlated with the strain-specific serological reaction. The presence or absence of plasmid DNA did not alter the strain-specific serological reaction or the DNA cleavage patterns. Both the serological reaction and the restriction enzyme digestion of total DNA were complementary to each other. These methods were used successfully to identify A. radiobacter K84 strains which were recovered 6 months after being inoculated to young trees in the field.     

Tinea capitis in the adult: Two case studies

Ascites fluid from hybridoma cell lines producing monoclonal antibodies to an exoantigen of Microsporum canis was assayed for its precipitating properties. Three types of monoclonal antibodies were used for serological analysis of heterologous mycelial cultures. Based on specific antigenic determinants, the fungal strains showed different immunodiffusion reactivity within the species. The monoclonal antibodies also proved to be effective reagents for serotyping heterogeneous species. They may also have potential applications in epidemiology.     

Immunochemical Characterization of Cell Wall Protein Antigen Purified from the Cell Wall Autolysate of Clostridium botulinum Type A

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components on immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Immunologic Analysis by Gel Diffusion of Effects of Prolonged Cultivation on Histomonas meleagridis (Smith)*

SYNOPSIS. Antigens were prepared from each of 4 lines of Histomonas meleagridis: Hm-L1, a strain highly virulent for both turkeys and chickens; Hm-L1 /C12, Hm-L1 /C24, Hm-L1 /C52, 3 avirulent substrains derived from Hm-L1 after 12, 24, 52 weeks of in vitro cultivation, respectively. Hm-L1 strain and the 3 substrains were maintained in liquid nitrogen. Antisera were developed in rabbits against Hm-L1 and Hm-L1 /C24 parasites. Both antisera were reacted on gel diffusion plates with homologous and heterologous antigens. Two groups of precipitin lines and/or bands designated arbitrarily as A and B, were observed on the slides. Analysis of these bands revealed the common antigenic composition of the 4 histomonads with respect to some of the group A and group B antigens. The concentrations and numbers of precipitin lines in both groups increased, however, with the length of cultivation. These antigenic differences are discussed in the light of their possible relationship to pathogenicity.     

Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 g protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 g gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.     

Detailed Assessment of the Kinetics of Hg-Cell Association,Hg Methylation,and Methylmercury Degradation in Several Desulfovibrio Species

The kinetics of inorganic Hg [Hg(II)_i] association, methylation, and methylmercury (MeHg) demethylation were examined for a group of Desulfovibrio species with and without MeHg production capability. We employed a detailed method for assessing MeHg production in cultures, including careful control of medium chemistry, cell density, and growth phase, plus mass balance of Hg(II)_i and MeHg during the assays. We tested the hypothesis that differences in Hg(II)_i sorption and/or uptake rates drive observed differences in methylation rates among Desulfovibrio species. Hg(II)_i associated rapidly and with high affinity to both methylating and nonmethylating species. MeHg production by Hg-methylating strains was rapid, plateauing after ∼3 h. All MeHg produced was rapidly exported. We also tested the idea that all Desulfovibrio species are capable of Hg(II)_i methylation but that rapid demethylation masks its production, but we found this was not the case. Therefore, the underlying reason why MeHg production capability is not universal in the Desulfovibrio is not differences in Hg affinity for cells nor differences in the ability of strains to degrade MeHg. However, Hg methylation rates varied substantially between Hg-methylating Desulfovibrio species even in these controlled experiments and after normalization to cell density. Thus, biological differences may drive cross-species differences in Hg methylation rates. As part of this study, we identified four new Hg methylators (Desulfovibrio aespoeensis, D. alkalitolerans, D. psychrotolerans, and D. sulfodismutans) and four nonmethylating species (Desulfovibrio alcoholivorans, D. tunisiensis, D. carbinoliphilus, and D. piger) in our ongoing effort to generate a library of strains for Hg methylation genomics.     

A Comparative Study of 14 Strains of Naegleria australiensis Demonstrates the Existence of a Highly Virulent Subspecies: N. australiensis italica n. spp.1

Fourteen strains of Naegleria australiensis, including the type strain, were compared for virulence for mice, maximum growth temperature, lectin agglutination, isoenzyme pattern, and total protein banding pattern. Their relation to other species of Naegleria also was compared by immunoelectrophoretic analysis. Strains with high virulence, comparable to that of N. fowleri, were found to be different in concanavalin A agglutination as well as with regard to zymograms and total protein patterns. Although serologically different from N. fowleri and reacting with N. australiensis antiserum in the fluorescent antibody test, these high-virulence strains differed in number of immunoelectrophoretic precipitin bands. Because of these results, the high-virulence strains are considered to be a subspecies of N. australiensis. The low-virulence strains showed minor differences from the type strain. Thus, N. australiensis does not appear to be as homogenous a species as N. fowleri. Pathogenic N. australiensis also seems to be more widespread than previously thought.     

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.     

Comparison of a Newly Developed Latex Agglutination Test and an Immunodiffusion Test in the Diagnosis of Systemic Candidiasis

A newly developed latex agglutination (LA) test and a modified immunodiffusion (ID) test were evaluated. The antigen used was a homogenate of Candida albicans. A total of 167 antisera were employed in the evaluation. They included 36 sera from clinically well persons; 78 from patients with various clinical forms of candidiasis; 52 from patients with proven cases of aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis, histoplasmosis, nocardiosis, paracoccidioidomycosis, sporotrichosis, and tuberculosis; and one serum from a patient with toruloposis. Use of the LA test in conjunction with the ID test permitted the detection of more than 90% of 43 proven candidiasis cases. Of all the heterologous cases and normal human sera tested, LA reactions were noted with 3 of 10 cryptococcosis case specimens, 1 of 9 tuberculosis case specimens, and with the torulopsemia case serum. In contrast, the only heterologous serum reactive in the ID test was that from the patient with torulopsemia. Torulopsis glabrata and C. albicans antisera gave identical reactions in LA and ID tests with T. glabrata or C. albicans antigens. ID tests with selected antigens, however, permitted differentiation of rabbit and human T. glabrata antibody from that of C. albicans antibody. Six different precipitins were recognized with the C. albicans antigens. The occurrence of multiple precipitin lines and high LA titers was suggestive of severe candidiasis. The LA test, in contrast to the ID test, appeared to have prognostic value. Together, the LA and ID tests provided a simple, rapid, and accurate means of detecting and monitoring infections by species of Candida.     

Nonvariant Antigens Limited to Bloodstream Forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense1

ABSTRACT. The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and Immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled ～3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.