Comparative Serology of the Marine Fish Pathogen Vibrio anguillarum

The different serotyping systems, based on thermostable O antigens, reported for Vibrio anguillarum and V. ordalii were compared by quantitative agglutination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent silver staining or Western blotting (immunoblotting) of purified lipopolysaccharide (LPS), using polyclonal rabbit antisera. The results demonstrate that 16 different serotypes within V. anguillarum (designated O1 to O16) can be distinguished. Each of these serotypes is characterized by a distinct polysaccharide banding pattern, as revealed by silver-stained gels of purified LPS. The comparative analysis allowed a complete alignment of the different serotypes for the first three serovars: O1, O2, and O3. Moreover, immunoblotting showed that strains belonging to each of these serotypes had the same LPS banding pattern independent of the origin of the strain. Serotype O2 contains different subtypes, O2a and O2b. While no differences were apparent between these subgroups in silver-stained gels, they could be separated by quantitative agglutination (titer determination) or immunoblotting. V. ordalii, the former biotype II of V. anguillarum, strongly reacts with anti-V. anguillarum O2a antiserum. Strains of the two species can be separated on the basis of different LPS profiles in the high-molecular-weight region of silver-stained gels of purified LPS. The silver-stained LPS profiles of the different serotypes of V. anguillarum that have been established are provided for further comparison in the future.     

The Use of Polyvalent Sera for the Serotyping of Mycobacteria within the Mycobacterium avium– M. intracellulare-M. scrofulaceum Complex

A modification to Schaefer's agglutination method for serotyping mycobacteria within the Mycobacterium avium-M. intracellulare-M. scrofulaceum complex is described. The antigens are screened against polyvalent sera and subsequently a reduced range of absorbed antisera. This expedites the serotyping procedure and conserves expensive antiserum stocks.     

Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae

Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens.     

Lipopolysaccharide heterogeneity in strains ofSerratia marcescens agglutinated by O14 antiserum

Lipopolysaccharides (LPS) from 71 strains ofSerratia marcescens that were agglutinated by O14 antiserum were examined by SDS-PAGE. Four major profiles were found, designated LPS1 to LPS4. These groups accounted for 51, 7, 5, and 3 strains respectively. Five strains were unclassified. Immunoblotting showed that O14 antibodies bound only to LPS1 and not to LPS2, 3, or 4. LPS1 also bound antibodies in O1, O4, O12, and O23 antisera. LPS2 reacted specifically with O8 antiserum, LPS3 with O6, and LPS4 with O2, O3, O6, O12, and O21 antisera. These reactions were not found in agglutination tests with boiled, whole-cell antigens. However, tests with autoclaved antigens (45 min at 121°C) corroborated the immunoblotting classifications; LPS1 strains belonged to serotype O14, LPS2 to serotype O8, LPS3 to serotype O6, and LPS4 to serotype O21. We conclude that there is a heat-stable antigen on many clinical strains ofS. marcescens that masks the expression of O-specific LPS antigens and which binds with nonspecific antibody in serum O14. We propose that O-antigens should be prepared from autoclaved cultures and that the H-reference strain O14H9 CDC 1783-57 (LPS2) should be reclassified as serotype O8.     

Serological Studies on Actinobacillus actinomycetem-comitans

One hundred strains of Actinobacillus actinomycetem-comitans were examined serologically by determining their agglutination reactions with six absorbed antisera. Twenty-four reaction patterns were found, and the results were reproducible. The agglutinating antigens were heat-stable. Precipitation reactions were also studied by means of the Ouchterlony agar-gel diffusion technique with Fuller and acetone extracts. The agglutination test was considered preferable for epidemiological and ecological investigations on A. actinomycetem-comitans.     

Development of an O-antigen serotyping scheme for Cronobacter sakazakii

Cronobacter sakazakii is an opportunistic pathogen that can cause severe infections. Serotyping provides a basis for the categorization of bacterial strains and is an important tool for epidemiological and surveillance purposes. In this study, of the 135 Cronobacter strains tested initially, 119 were identified as C. sakazakii and used. A serotyping scheme for C. sakazakii that classifies strains based on their different O antigens was developed. Seven antisera that exhibited high agglutinin titers (>640) were produced. O2 and O6 antisera were specific for their homologous strains, O4 and O7 antisera gave heterologous titers with O1 and O6 antigens, respectively, and O1, O3, and O5 antisera cross-reacted with each other and require preabsorption with the other two antigens. All of these 119 C. sakazakii strains were clearly assigned to these seven serotypes. O1 and O2 are the dominant serotypes, comprising 69.7% of the isolates. We also characterized the O-antigen gene clusters using restriction fragment length polymorphism (RFLP). The grouping of C. sakazakii strains based on their RFLP banding patterns correlated well with the grouping of strains based on our serotyping scheme. The serotype scheme presented here could prove to be a useful tool for serotyping C. sakazakii isolates.     

Immunological characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test and reactivity with patient sera of tsutsugamushi diseases.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Penner serotyping of Campylobacter isolates from poultry, with absorbed pooled antisera

The Penner serotyping system, based on detection of heat-stable antigens with a passive haemagglutination technique, was used in studies on Campylobacter epidemiology in poultry. Preparation of specific antisera by absorption allowed the use of pooled antisera. Over 80% of the Campylobacter isolates were typable with this modified Penner serotyping system. Typability of strains was clearly affected by storage of the strains before actual typing. Extracted antigens appeared to be stable for at least 6 months at 4°C. Therefore, it is advisable to store extracted antigens from freshly isolated Campylobacter strains instead of reculturing frozen-stored strains, when actual typing cannot be performed directly after primary isolation. Untypability of isolates may partly be explained by the detection of Campylobacter serovars not yet represented in the serotyping system. Experiments on repeated serotyping of several Campylobacter strains did not suggest any serovar instability within the strains.     

Detection of type-specific antibody to lymphogranuloma venereum serotypes ofChlamydia trachomatis with a microneutralization test

Ten-day monospecific mouse antisera prepared against serotypes L1, L2, and L3 ofChlamydia trachomatis were titrated against homologous and heterologous antigens in the microimmunofluorescence (micro-IF) test and by a new microneutralization method. While broad crossreactivity between serotypes was detected in the micro-IF test, the same antisera demonstrated serotype specific neutralizing activity. The microneutralization test could be used for serotyping new chlamydial isolates when equivocal results have been obtained by the microimmunofluorescence method.     

Some serological properties ofActinobacillus pleuropneumoniae strains of serotypes 1 through 5

Rabbits were hyperimmunized with live, formalin-killed, and heat-treated antigen preparations of the reference strains of serotypes 1 through 5 ofActinobacillus pleuropneumoniae in order to study the antibody response to both soluble and particulate antigens. The antibody response was studied by means of precipitation, agglutination, coagglutination, indirect hemagglutination, and complement fixation tests.Serotyping ofA. pleuropneumoniae strains was done by ring precipitation (RP) and coagglutination (CoA) tests with unheated and heated cell-saline extract as antigens and rabbit hyperimmune sera produced against either live cultures or formalin-killed whole-cell suspensions. The results showed that live cultures provoked more cross-reactive antibodies in rabbits, thus making the antisera unsuitable for use in serotyping by the RP test when unheated wholecell saline extract was used as antigen. Rabbit hyperimmune serum produced against formalinkilled bacterial suspension gave serotype-specific reactions in the RP test. Boiled or autoclaved cell-saline extracts gave serotype-specific reactions in the RP test even when rabbit anti-livecell sera were used. Serotype-specific reactions were obtained in the CoA test in both rabbit anti-live or anti-formalin-killed cell sera with either unheated or heated bacterial cell suspensions as antigens.Live and formalin-killed whole-cell suspensions as well as their saline extracts provoked a high antibody response in rabbits. Heating the cell suspension at 100°C for 1 h caused a significant reduction in their immunogenic potency, whereas autoclaving (121°C) of the cell suspension for 1 h almost completely destroyed their serotype-specific immunogenic properties, since the antibody response was either absent or very poor and not type-specific. However, neither boiling nor autoclaving of the cell suspensions caused significant reduction in their ability to react with preformed antibodies. Phenol-water-extracted antigens gave the highest degree of serotype specificity in the complement fixation test.     

Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, O9) of Salmonella typhi

A panel of monoclonal antibodies were generated against the surface polysaccharide antigens of the cell envelope of Salmonella typhi. Four clones (IgM) were specific for the capsular Vi polysaccharide, and one clone (IgG3) reacted selectively with the S. typhi lipopolysaccharide in enzyme immunoassay. On the basis of their reactivity pattern and binding affinity, MATy-V7 (IgM) and MATy-O9 (IgG3) antibodies were selected for further characterization of their antigenic specificity. In an inhibition enzyme immunoassay with rabbit factor-specific anti-Salmonella antibodies as the competing agents, the reactivity of MATy-V7 and MATy-O9 were significantly inhibited by the anti-Vi and anti-O9 antisera, respectively. Moreover, both the Vi- and O9-specific monoclonal antibodies were shown to be useful serotyping agents by correct identification in slide agglutination tests of 32 clinical isolates of all the S. typhi and other serogroup D salmonellae among a total of 140 bacterial isolates representing eight different Enterobacteriaceae genera tested.     

Serological Specificity of the Lipopolysaccharides, the Major Antigens of Pseudomonas syringae

The nature of major antigens of Pseudomonas syringae was studied on one strain of four pathovars (pvs aptata, mors-prunorum, phaseolicola and tabaci) belonging to four separate serogroups. Bacterial antigens were prepared by 4 procedures: extraction by phenol-water (PW), by citrate-NaCl (CN), by trichloracetic acid (TCA), and precipitation of a glycoproteic extracellular complex (GP). 3-Deoxy-2-octulosonic acid (KDO) revelation in all the extracts showed that the four procedures led to antigens containing similar amounts of lipopolysaccharide (LPS). Twenty polyclonal antisera were raised in rabbits against whole bacteria and the different extracts. Serological reactions were tested by gel double diffusion (DD) and indirect immunofluorescent staining (IF). The anti-whole cell sera were shown to contain mostly anti-LPS antibodies. For each pathovar, whole bacteria used as antigens in DD gave precipitation bands identical to the bands given by the LPS extracts (PW, CN or TCA), identical to the heated bacteria (HB), and identical to LPS sidechain preparations. The GP extract itself was shown to be rich in LPS. To serotype P. syringae, it is advised to raise antisera against either whole bacteria or GP extracts; whereas the reacting antigens for DD would be heated bacteria.     

Heat-stable serotyping antigens expressed by strains of Campylobacter jejuni are probably capsular and not long-chain lipopolysaccharide

The role of lipopolysaccharide (LPS) in the serotyping of Campylobacter jejuni based on heat-stable antigens was examined using SDS-PAGE and a silver stain for carbohydrate. None of the 32 type strains of Camp. jejuni expressed long-chain LPS. Rabbit antibodies, prepared to 10 selected strains of Camp. jejuni , reacted with surface-exposed carbohydrate antigens, which were not LPS. This study suggests that the heat-stable antigens of Camp. jejuni , which form the basis for the established Penner serotyping scheme, are probably capsular and not LPS.     

Isolation from Pasteurella multocida of a Lipopolysaccharide Antigen with Immunizing and Toxic Properties

A heat-stable, particulate, lipopolysaccharide-protein antigenic complex has been isolated from a virulent, encapsulated strain of Pasteurella multocida by extraction with cold, formalinized saline, and centrifugation at 105,000 x g. The original bacterial culture had been obtained from a bison that died of hemorrhagic septicemia, an infectious disease of cattle and buffalo. Injection of fractional milligram amounts of the antigen into mice, rabbits, and calves produced toxic reactions which frequently resulted in death of the host. The surviving animals demonstrated a high degree of immunity to challenge with live, virulent organisms. Two injections with 15 mug of the antigen produced a high degree of immunity in mice without the development of any signs of toxicity. The gross chemical composition and toxicity of the antigen were similar to those reported for endotoxins obtained by the Boivin or Westphal procedure. Although strong serological cross-reactions were obtained in Ouchterlony plates between the 105,000 x g antigens from the bison strain and an avian strain with antisera to these strains, these antisera agglutinated only the bacterial cells of the homologous strain. The active immunity obtained in mice by the injection of the 105,000 x g antigens of each strain was specific and could be correlated with the agglutination tests.     

Hemagglutination Inhibition for Serogrouping of Neisseria meningitidis

A hemagglutination inhibition (HI) test has been studied as an alternative to bacterial agglutination (BA) for serogrouping strains of Neisseria meningitidis isolated from clinical specimens. The HI test consists of polysaccharide antigens adsorbed to sheep red blood cells which were then agglutinated by group-specific antisera. Supernatant fluids from suspensions of meningococci were used to inhibit the agglutination. Results of the two tests agreed for 381 (80%) carrier strains. Of the remaining 95 strains, 82 (86%) were identified by HI although they were nongroupable by BA. Thus, the HI test has been shown to be more highly specific and sensitive and to be more economical of reagents and time than the BA test.     

Antigenic determinants and specificity of antisera against acidophilic bacteria

Polyclonal antisera, produced against whole cells of Thiobacillus thiooxidans, T. ferrooxidans and Leptospirillum ferrooxidans, gave highly specific reactions when cross-reacted with 23 strains of acidophilic bacteria using an immunofluorescence (IF) staining technique. Strains of identical serotype exhibited maximum cross-reaction whereas strains of different serotype reacted only weakly. Lipopolysaccharides (LPS) examined by SDS-PAGE showed different, serotype-specific migration patterns indicating their rough or smooth character. LPS patterns may therefore be used for serological classification of acidophilic bacteria. Surface antigens of four strains were identified by immunoblot staining; LPS and some proteins were antigenic determinants with LPS the most specific.The authors are with the Universität Hamburg, Institut für Allgemeine Botanik, Abteilung für Mikrobiologie, Ohnhorststrasse 18, D-22609 Hamburg, Germany     

Antigenic Relationships Among Strains of Vibrio parahaemolyticus

The antigenic relationships of 79 strains of Vibrio parahaemolyticus representing 46 assigned K-types were studied by tube agglutination. Homologous titers of 46 anti-K sera ranged from 80 to 2,560. All but three sera exhibited from one to six heterologous reactions, the majority of which gave titers of 相似文献   

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K,D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.