Differentiation of the major flagellar antigens of Pseudomonas aeruginosa by the slide coagglutination technique.

Antisera against the two major flagellar antigens of Pseudomonas aeruginosa were obtained by immunization of rabbits with isolated flagella and absorption of contaminating antisomatic antibodies. In the conventional slide agglutination test, the pure H antisera did not agglutinate the flagellated cells of the homologous strains. The addition of protein A-bearing staphylococci to H antiserum and homologous flagellated cells, the so-called slide coagglutination, results in a rapid development of flaky clumps. H coagglutination tests of reference strains, which formerly have been H typed by long-term tube agglutination and by the indirect fluorescent-antibody technique, yielded exactly the same subdivision of the strains in H type a and H type b as the more laborious and time-consuming methods. O grouping and H typing of 181 isolates from clinical specimens revealed a free combination of the somatic and flagellar antigens. 25 OH serovars were found. The simple and rapid coagglutination technique can promote the serovar determination of P. aeruginosa, particularly for the purpose of hospital infection control.     

Evaluation of commercial antisera for Salmonella serotyping

We compared a set of commercial Salmonella somatic and flagellar serotyping antisera to in-house-prepared antisera from the Microbial Diseases Laboratory, California Department of Public Health, using 327 Salmonella enterica strains belonging to subgroups I, II, IIIa, IIIb, and IV. The sensitivities of Denka Seiken (Tokyo, Japan) somatic and flagellar antisera (using a tube agglutination assay) were 94.0% and 99.2%, respectively, and the specificity was 100% for both sets of sera. Polyvalent O and O1 antiserum sensitivity and specificity were >90%, with the exception of polyvalent O1 antiserum, for which sensitivity was 88.9%. When Denka Seiken flagellar antisera were used in a slide agglutination assay, the sensitivity and accuracy dropped to 88.9% and the specificity fell to 91%. Overall, Denka Seiken commercial antisera performed very well and, together with the comprehensive range of factors available, offer laboratories quality reagents suitable for serotyping strains of salmonellae.     

Vibrio cholerae flagellar antigens: A serodiagnostic test,functional implications of H-reactivity and taxonomic importance of cross-reactions within theVibrio genus

Serodiagnostic tests for all serotypes of Vibrio cholerae using H-antisera were investigated. Activity motile cell lines of 155 stock and international reference cultures of human, animal, fish, and halophilic Vibrios, Aeromonas, Comomonas, Pseudomonas, Salmonella, and Escherichia were investigated. Without exception, all cholera vibrios (including the NAG serotypes) reacted with H sera. Positive reactions were obtained specifically (a) within 2 hrs at 52 degrees C in the tube test using thick formalized suspensions and H antisera at optimal proportion titre and (b) within 30 sec by slide agglutination of fresh cultures. The other vibrios investigated reacted similarly with their homologous H antisera. 2. The rapid diagnostic techniques of fluorescent antibody labeling or immobilization were unsuccessful, V. cholerae flagella being refractive to H sera in these tests. V. cholerae was, however, sensitive in a type-specific manner to O antisera. These and related observations suggest that O antigen has a functional role in Vibrio motility. 3. Interspecies H cross-reactions between V. cholerae and fish and animal vibrios which correlated with bacteriologic similarity, were demonstrated. O antigens of these vibrios were strain specific. Cross-absorption analysis indicated that the H antigens of vibrios were characteristic and homogenous within the species, and therefore a potentially important taxonomic criterion of Vibrio species.     

Serological grouping of streptococci by slide agglutination.

Streptococcal grouping sera for groups A, B, C, and G prepared for conventional testing by precipitation were made specific by absorption and used to identify streptococci by slide agglutination with and without staphyloccocal coagglutination. Trypsinised suspensions of 1055 strains, identified by precipitation as belonging to group A, B, C, or G, were tested by slide agglutination. Of these, 998 were correctly identified using a streptococcal suspension and antisera alone and a further 65 were identified when a loopful of protein A-positive staphylococci was added. Suspensions of 88 strains not of groups A, B, C, or G gave no reaction in the agglutination test with or without the addition of staphylococci. Group polysaccharide extracted by conventional methods also caused agglutination of staphylococci on a slide when specific antiserum was added. Growth from primary or secondary cultures digested in streptomyces enzyme for only 15-30 minutes provided an excellent antigen for a quick and simple method of streptococcal grouping using non-sensitised staphylococcal suspension and specific antisera for coagglutination.     

Typing of Haemophilus influenzae by Coagglutination and Conventional Slide Agglutination

Coagglutination was compared with conventional slide agglutination for the typing of 297 clinical isolates of Haemophilus sp. A 100% correlation was found with the H. influenzae type b isolates. Coagglutination showed no false-positive reactions with the nontypable strains of H. influenzae and H. parainfluenzae isolates; however, conventional slide agglutination exhibited many false-positive and non-interpretable reactions.     

Comparison of methods for serotyping isolates of Haemophilus influenzae.

This study compared four methods for serotyping isolates of Haemophilus influenzae. Slide agglutination with commercial antisera (Difco Laboratories and Wellcome Diagnostics), coagglutination (Phadebact Haemophilus Test [Pharmacia Fine Chemicals]), latex agglutination with affinity-purified anticapsular antibody, and counterimmunoelectrophoresis with multiple antisera were used to serotype 80 isolates of H. influenzae. Coagglutination and counterimmunoelectrophoresis correctly identified all 80 isolates as either type b or not type b. Slide agglutination and latex agglutination each successfully identified 76 of the 80 isolates; however, each of these two methods failed to type four isolates because of agglutination of controls. We recommend slide agglutination or coagglutination as the serotyping methods of choice in most laboratories because they are simple, accurate, and rapid. Slide agglutination with Difco antiserum can be performed at the lowest cost.     

Evaluation of commercial latex reagents for identification of O157 and H7 antigens of Escherichia coli.

Agglutination reactions obtained with three commercial latex reagents for detecting Escherichia coli O157 antigen (Oxoid Diagnostic Reagents, Hampshire, England; Pro-Lab Inc., Richmond Hill, Ontario, Canada; and Remel Microbiology Products, Lenexa, Kans.) and one for detecting H7 antigen (Remel) were compared with those obtained by standard serologic methods by using the Centers for Disease Control and Prevention (CDC) reference antisera for O157 and H7 antigens. For 159 strains of E. coli and related organisms, the Oxoid, Pro-Lab, and Remel O157 latex reagents each had a sensitivity and specificity of 100% compared with the CDC reference antiserum. For 106 strains of E. coli and related organisms that were not enhanced for motility through semisolid medium, the Remel H7 latex reagent had a sensitivity of 96% and a specificity of 100% compared with the standard tube agglutination method with CDC H7 antiserum. Measures to enhance motility were needed for some strains to detect the H7 antigen. Our findings demonstrate that the commercial latex reagents are good alternatives to standard serologic methods for identifying the O157 and H7 antigens of E. coli.     

Rapid slide coagglutination test for identifying and typing group B streptococci.

A Cowan I strain of Staphylococcus aureus was labeled with either group B streptococcal grouping or typing antiserum. These antibody-labeled reagent cells (ARC) were used in a slide coagglutination test to identify and type group B streptococci from blood agar plates. All streptococci were also identified by the standard Lancefield capillary precipitin test. In a blind study, all 141 group B streptococci were correctly identified by the coagglutination grouping test. None of the 148 non-group B streptococci caused agglutination of ARC. The coagglutination grouping test required an acid extract prepared from only four colonies and could be completed less than 30 min after colonies were removed from plates. The coagglutination typing test correctly identified 98.6% of the types of the 141 group B streptococcal strains tested. At least 88.6% of these streptococci could be typed directly from blood agar plates within 5 min by the coagglutination typing test. The remaining 11.4% of the group B streptococci were acid extracted (less than a 30-min procedure), and the extract was used for coagglutination typing. Coagglutination typing can be performed with only four colonies. The coagglutination grouping and typing tests are inexpensive, rapid, reliable, and easy to perform.     

Comparison of indirect hemagglutination and rapid plate agglutination tests with counterimmunoelectrophoresis for typing Pasteurella haemolytica.

A rapid, simple, and accurate counterimmunoelectrophoresis (CIE) technique was developed and compared with the indirect hemagglutination and rapid plate agglutination techniques for serotyping cultures of Pasteurella haemolytica. The CIE test had 100% correlation with the conventional indirect hemagglutination test and, after serum absorption, correctly identified cultures representing the 12 established serotypes and 49 field isolates of P. haemolytica with reasonable rapidity. Cross-reactions were observed in the CIE and rapid plate agglutination tests but not in the indirect hemagglutination test with antisera prepared from the 12 established serotypes. These cross-reactions were eliminated from the CIE test but not from the rapid plate agglutination test by absorption of antisera with cells which possessed the cross-reacting antigens. Avian isolates of P. haemolytica did not type with antisera to the 12 established serotypes by any of the methods. Both homologous and heterologous reactions were observed with these strains in the rapid plate agglutination and CIE tests with antisera prepared from six selected cultures. These results support the previous finding that the taxonomic relationship of these avian strains to P. haemolytica is questionable.     

O and H serotyping of Pseudomonas cepacia.

Procedures for the preparation, absorption, and titration of Pseudomonas cepacia O and H rabbit antisera are described. Seven O antigens (O1 to O7) for the slide agglutination test and five H antigens (H1, H3, H5, H6, and H7) for the agglutination and H immobilization tests were determined. Nearly 300 strains of P. cepacia isolated from hospitalized patients (a majority from Strasbourg hospitals) were serotyped. The use of P. cepacia serotyping as an epidemiological tool, especially in outbreak situations, was emphasized. Difficulties in obtaining monospecific H antisera are discussed.     

Quality of commercially produced Shigella serogrouping and serotyping antisera.

Shigella grouping antisera from five manufacturers and typing antisera from two were purchased and evaluated with homologous and heterologous Shigella strains in the slide agglutination test. Only 31 of 73 (42%) antisera were satisfactory. In many instances, the antisera gave negative, as opposed to weak, reactions when they should have given strong positive reactions. Four reagents cross-reacted with Shigella strains. Of the 19 polyvalent grouping antisera to subgroups Shigella dysenteriae serotypes 1 through 7, S. flexneri serotypes 1 through 6, S. boydii serotypes 1 through 7, and S. sonnei forms I, II, only one S. sonnei reagent and five S. flexneri reagents were satisfactory with greater than or equal to 90% of the homologous strains. The reagent of poorest quality was satisfactory with only 18% of the homologous strains. There were three polyvalent antisera to the higher types of S. dysenteriae and S. boydii, which were available from only one company, that adequately identified 80, 63, and 65% of the homologous strains. Typing antisera were available from only two companies, and 30 of 51 (59%) were satisfactory. Commercially available Shigella antisera are inadequate for the laboratory testing required for planning the development of and evaluating Shigella vaccines.     

Methods for serotyping nasopharyngeal isolates of Haemophilus influenzae: slide agglutination, Quellung reaction, countercurrent immunoelectrophoresis, latex agglutination, and antiserum agar.

Nasopharyngeal isolates of H. influenzae were typed by the slide agglutination test, the Quelling reaction, the latex agglutination test, countercurrent immunoelectrophoresis, and the antiserum agar test. These tests gave essentially comparable results, with countercurrent immunoelectrophoresis and latex agglutination being slightly more sensitive. Cross-reactive problems encountered with latex agglutination and the expense of performing countercurrent immunoelectrophoresis or the antiserum agar test made these tests less practical than the slide agglutination test to identify single strains that were already isolated. The Quellung reaction and slide agglutination were the most rapid tests used to type an organism. For mass screening of multiple samples, countercurrent immunoelectrophoresis was the simplest technique. The antiserum agar test was slow but was the best technique to screen nasopharyngeal swab cultures to identify the presence of any encapsulated strains in the mixed flora. Whether any of the above techniques were as sensitive as the immunofluorescence test was not evaluated in this study.     

Fluorescent-antibody studies on selected strains of Bacteroides fragilis subspecies fragilis.

Antisera against seven strains of Bacteroides fragilis subspecies fragilis were produced from dense suspensions of whole cells. These sera exhibited high agglutination titers with homologous antigens. Reciprocal cross-reactions in agglutination tests with each immunizing strain yielded lower titers. Both the indirect and direct fluorescent-antibody techniques were used to evaluate these reagents in the serological identification of 24 defined strains of B. fragilis subspecies fragilis. Subspecies and even strain specificities were noted with particular antisera. A pooled antiserum and conjugate were prepared and studied. Study results showed that specific and high-titered antisera against strains within this subspecies can be produced by the methods described herein and that possibly more than one serotype exists within the seven strains studied. The development of more antibody pools will be necessary to encompass a wider antigenic coverage before the fluorescent-antibody technique can be relied upon altogether for serologically identifying isolates of B. fragilis subspecies fragilis. Test data showed that the indirect method of fluorescent-antibody staining with whole antiserum is an excellent means of identifying strains of this organism.     

Relationship of Serogroups of Neisseria meningitidis I. Microagglutination, Gel Diffusion, and Slide Agglutination Studies of Meningococcal Antisera Before and After Absorption with RAS-10 Strain of Meningococci

Microagglutination tests were used to show the relationship of a nongroupable strain of Neisseria meningitidis (RAS-10) to other serological groups. RAS-10 antiserum has been prepared and studied for the first time. Antibodies to the RAS-10 strain were shown to be present in many grouping antisera obtained from different sources. These antibodies were absorbed from antisera to heterologous sero-groups with the RAS-10 strain. This procedure was shown to make antisera more specific by eliminating serological cross-reactions and false grouping of RAS-10 strains. Antisera before and after absorption with RAS-10 cells were studied by using double diffusion in gels. An antigen-antibody precipitation line for the RAS-10 meningococci was shown to be removed by this procedure. Antiserum to group 29E meningococci was absorbed with group Z cells, and precipitation lines for Z cells were removed. Group 29E antiserum agglutinated group 29E and group Z cells in the slide agglutination test but was specific for group 29E cells in this test after absorption with group Z cells.     

Comparison of slide agglutination test and direct immunofluorescence assay for identification of Legionella isolates.

It is technically impractical for many clinical laboratories to use the direct immunofluorescence assay for identifying and serogrouping clinical isolates of Legionella. We compared the results obtained with the direct immunofluorescence assay with the results of a simple and less-demanding slide agglutination test for identifying 15 serogroups representing seven Legionella species. The slide agglutination test was in complete agreement with the direct immunofluorescence assay, and the serogroup to which 64 clinical isolates of Legionella belonged was correctly identified. With polyvalent, pooled antisera and absorbed, serogroup-specific antisera, the slide agglutination test is a useful alternative to the direct immunofluorescence assay in the diagnosis of Legionella infections and for studying the serological relationships of Legionella-like organisms.     

Monoclonal antibody-based enzyme-linked immunosorbent assays for identification and serotyping of Vibrio cholerae O1.

Monoclonal antibodies directed against O-specific antigens of Vibrio cholerae O1 lipopolysaccharide were used in two different enzyme-linked immunosorbent assays (ELISAs), designed for identification and serotyping of V. cholerae O1. In the sandwich ELISA, a monoclonal antibody against the group-specific antigen was used as capture antibody, whereas peroxidase-conjugated monoclonal antibodies directed against group- and type-specific antigens were used as the second antibodies. Monoclonal antibodies were also used in ELISA inhibition tests with whole bacteria as inhibitors in microtiter trays coated with V. cholerae O1 lipopolysaccharide. In addition, the monoclonal antibodies were shown to be useful in slide agglutination tests. The enzyme immunoassays were equally sensitive, showing positive reactions with all V. cholerae O1 strains tested, whereas all V. cholerae non-O1 as well as strains of Escherichia coli, Shigella sonnei, Salmonella spp., Citrobacter freundii, and Brucella abortus were negative. The microtiter application makes the immunoassays suitable with low consumption of reagents for screening of samples from suspected cases as well as from the environment.     

Serotyping of Listeria monocytogenes by enzyme-linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting

Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of L. monocytogenes antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of L. monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.     

Characterization of murine monoclonal antibodies against serogroup B salmonellae and application as serotyping reagents.

Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other O serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory.     

Identification of 22 Legionella species and 33 serogroups with the slide agglutination test.

We used the slide agglutination test to determine the serologic relationships of 22 Legionella spp. representing 33 serogroups. Antisera prepared against 14 of the Legionella spp. contained cross-reactive antibodies (1+ or greater) at their working dilutions. Numerous cross-reactions were observed for the blue-white fluorescing Legionella spp. With only three exceptions in the latter group, cross-reactive antibodies were removed by absorption, thereby producing serogroup-specific antisera. For screening tests or for identification only to the genus level, nine polyvalent antiserum pools were prepared. Routine use of slide agglutination test reagents should expand the number of Legionella spp. that can be identified in the clinical laboratory and, at the same time, provide a simpler, less costly test procedure.     

Serotyping of Clostridium difficile.

A total of 246 live Clostridium difficile cultures were serotyped by a slide agglutination technique. Fifteen grouping antisera were produced which serotyped 98% of the cultures (241 of 246). Our results indicated that certain serogroups may have specific pathogenicity. Strains of serogroups A, G, H, K, S1, and S4 were cytotoxigenic and were isolated mainly from adult patients with pseudomembranous colitis or antibiotic-associated diarrhea. Nontoxigenic strains of serogroups D and Cd-5 were isolated mainly from asymptomatic neonates and small children. Some cross-reactions occurred among some strains of serogroups A, Cd-5, G, and K. These strains were further examined by analysis of protein profiles and restriction endonuclease patterns to elucidate their serology. Typing of C. difficile by using slide agglutination is a simple technique suitable for routine examination. Serogrouping may be a useful epidemiological marker and could help in elucidating the medical relevance of some C. difficile isolates.