Cross-reactivity and antigenic heterogeneity among Actinobacillus pleuropneumoniae strains of serotypes 4 and 7.

Actinobacillus pleuropneumoniae strains of serotypes 4 and 7 were studied for their antigenic properties by means of agglutination, coagglutination, indirect hemagglutination, immunodiffusion, and counterimmunoelectrophoresis tests. Strains of serotype 4 showed cross-reactivity with those of serotype 7 in various serological tests. Serotype 7 strains were antigenically heterogeneous and shared common antigens with several other serotypes. By using boiled whole-cell saline extract as the antigen in the immunodiffusion test, serotype 7 strains could be divided into four subgroups. Subgroup I strains did not have antigens in common with other serotypes, whereas subgroup II strains had antigens in common with serotype 4; subgroup III strains had antigens in common with serotype 10, and subgroup IV had antigens in common with serotypes 1, 9, and 11. The indirect hemagglutination test using unheated whole-cell saline extract as the antigen detected serotype-specific activity. Quantification of serotype-specific and group-specific antigens by coagglutination and immunodiffusion tests was found useful for identifying strains that belonged to serotype 4 or 7.     

Cross-reactions between Actinobacillus (Haemophilus) pleuropneumoniae strains of serotypes 1 and 9.

Actinobacillus pleuropneumoniae strains of serotypes 1 and 9 were studied for their serological properties by means of agglutination, coagglutination (CoA), indirect hemagglutination (IHA), Co-IHA, ring precipitation (RP), and immunodiffusion (ID) tests. Particulate and soluble antigens of unheated and heat-treated bacterial cells were used in various serological tests. Agglutination, CoA, and RP tests demonstrated common antigens between strains of serotypes 1 and 9. Quantitative estimation of serotype-specific antigenic activity by CoA, RP, and ID tests proved useful in differentiating strains of serotypes 1 and 9. IHA and Co-IHA tests using sheep erythrocytes sensitized with unheated or heat-treated whole-cell saline extract and the ID test using boiled whole-cell saline extract as antigen distinguished the strains of serotypes 1 and 9. In studies of absorption of rabbit antisera with heterologous whole-cell antigens there was no absorption of antibodies in tube agglutination and IHA tests, suggesting that serotype 1 and 9 strains belong to two distinct serogroups. It appears that the cross-reactivity between serotype 1 and 9 strains could be due to common epitopes associated with cell wall antigens.     

Relationships between serological groups and deoxyribonucleic acid homology groups in Bacteroides fragilis and related species.

The serological properties of antigens extracted from strains of Bacteroides fragilis and related species belonging to several different deoxyribonucleic acid homology groups were investigated. Antisera prepared against Formalin-treated whole cell suspensions of representative strains were tested against cell suspensions, cell wall preparations, and extracts of homologous and heterologous strains by using agglutination, immunodiffusion, and hemagglutination techniques. Serological results indicated that the species were antigenically distinct, although minor cross-reactions were observed. Homology groups, including the two B. fragilis subgroups, were relatively homogeneous, although the presence of serotypes within each homology group was suggested. Immunodiffusion tests demonstrated, however, that each possessed a mosaic antigen composition; at least 6 antigenic determinants could be demonstrated in B. fragilis 2553, and up to 10 were found in B. fragilis 2393. Hemagglutination tests using antigen extracts also indicate a mosaic of antigens in each strain.     

Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease.

Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.     

Enzyme-linked immunosorbent assay for detection of specific antibodies to Ureaplasma urealyticum serotypes.

Optimal conditions of a micro-enzyme-linked immunosorbent assay system for the detection of immunoglobulin G antibodies to Ureaplasma urealyticum were established with rabbit antisera. Initially, the antisera, raised against eight U. urealyticum serotypes grown on medium containing horse serum, displayed nonspecific reactions with our enzyme-linked immunosorbent assay antigens. Substitution of fetal bovine serum in the medium eliminated this nonspecificity. The assay was then serotype-specific for the original eight U. urealyticum serotypes. The prominent homologous reaction was easily differentiated from the heterologous reactions. A one-way cross-reaction was observed with serotype 2 antiserum and serotype 5 antigen. The results were reproducible and could be obtained in 4 h with only 10 microliters of serum for eight serotypes. Optimal antigen concentrations for the U. urealyticum serotypes ranged from 0.40 to 1.60 micrograms/ml. Our results indicated that enzyme-linked immunosorbent assay has the potential for the detection of antibodies to specific serotypes of U. urealyticum.     

Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9.

The study described here was carried out to further characterize reference strains of Serpulina (Treponema) hyodysenteriae representing serotypes 8 and 9. Results obtained from restriction fragment length polymorphism analysis, enteropathogenicity testing, and endotoxin profiles confirmed their identifications. Electron microscopy indicated that both strains were covered with a thin layer of capsule-like material. Immunoblot analysis indicated that an antigen in the 19-kDa region of proteinase K-digested whole cells reacted only with homologous antiserum. The serotype-specific antigens were sensitive to periodate oxidation but resistant to proteinase K digestion and migrated in the same region as purified lipopolysaccharides. Immunoblotting with proteinase K-digested whole cells appeared as useful as immunodiffusion with extracted lipopolysaccharide for the serological classification of S. hyodysenteriae. Immunogold labeling of whole cells and purified periplasmic flagella showed strong cross-reactions between S. hyodysenteriae and Serpulina innocens. Outer membrane preparations of strains representing serotypes 8 and 9 contained four major proteins which reacted with antisera against both species, and one major protein with a molecular mass of 46 kDa which reacted only with antisera against S. hyodysenteriae, irrespective of the serotype. Our findings suggest that periplasmic flagella and some outer membrane proteins are antigens common to both S. hyodysenteriae and S. innocens, whereas a 46-kDa outer membrane protein may be a species-specific antigen of S. hyodysenteriae. Finally, we propose immunoblotting as an alternative method to immunodiffusion for the serotyping of S. hyodysenteriae.     

Broadly reacting precipitating and agglutinating antigen of leptospirae.

A saprophytic Leptospira biflexa strain of equine origin was found which cross-reacts with immune rabbit antisera to 14 pathogenic Leptospira interrogans serotypes. Sera from goats experimentally inoculated with the saprophyte showed multiple low-level cross-agglutination reactions against a battery of live L. interrogans serotypes. Sonically treated and saline-extracted suspensions of the L. biflexa strain and serotypes canicola, icterohaemorrhagiae, and pomona yielded a common precipitating protein antigen that was detected by immunodiffusion and immunoelectrophoresis with all of the antileptospiral sera examined. In cross-absorption and gel diffusion tests, the precipitinogen from each of the strains was shown to be identical. Formaldehyde treatments and heating at 100 degree C suggest that the cellular location of the common antigen is either somatic or subsurface, and Sephadex G-200 gel filtration enabled the isolation of the active fraction of the L. biflexa antigen. Monoprecipitin sera against the common antigen of L. biflexa were produced by immunizing rabbits with specific precipitates in agar. In gel diffusion and immunoelectrophoresis tests the antisera with each of the soluble antigens developed a single precipitin formation, and the antisera agglutinated formolized and heated whole-cell suspensions of serotypes canicola, icterohaemorrhagiae, and pomona at low dilutions. The soluble L. biflexa antigen was evaluated as an immunogen and in passive immunity tests for protection against death and kidney infection in hamsters. No cross-protection occurred when the hamsters were challenged with virulent leptospires. In contrast, the animals vaccinated or administered hamster immune serum before challenge died earlier than the control animals.     

Immunodominant Nocardia asteroides antigens: isolation and characterisation by enzyme-linked immunoelectrotransfer blotting, and the value of immunoblot strips.

Concentrated cell-free filtrates (nocardins) were prepared from Nocardia asteroides cultures grown on Sauton's synthetic broth. Nocardins from 10 strains of six N. asteroides serotypes were produced and the proteins separated by isoelectric focusing. N. asteroides antigens among these proteins were tested for specificity using rabbit antisera and monoclonal antibodies (MAbs) against N. asteroides and Mycobacterium tuberculosis by the enzyme-linked immunoelectrotransfer blot test. At least 15 protein antigens were identified from each of the 10 nocardins. The immunodominant antigens were one serotype-specific N. asteroides protein with an isoelectric point (pI) of 4.0 (factor 1) and two group antigens with pIs of 4.43 (factor 6) and 4.68 (factor 8). The nitrocellulose strips prepared with these antigens did not react with antibodies to M. tuberculosis, nor with normal sera from humans, rabbits, or mice, but reacted specifically with anti-N. asteroides MAbs and polyclonal antibodies. Four purified protein derivatives of tuberculin were tested and did not cross-react with the three anti-N. asteroides MAbs. These reactions suggest that the antigens identified as factors 1, 6 and 7 are specific to N. asteroides and that factor 1 is specific for serotype 2, while factors 6 and 8 are species-specific.     

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.     

The use of an enzyme-linked immunosorbent assay to detect IgG antibodies to serotype-specific and group-specific antigens of fowl adenovirus serotypes 2, 3 and 4

A sensitive enzyme-linked immunosorbent assay (ELISA) has been developed which can detect serotype and group-specific antibodies (IgG) to fowl adenovirus serotypes 2, 3 and 4. The chickens produced principally type-specific antibodies after a single oral inoculation of virus which enabled that strain to be identified by the ELISA. However, inoculation of an heterologous serotype, although inducing strain-specific antibodies to itself, also induced high levels of antibody to the group-specific antigens. This masked the serotype-specific antibodies in the ELISA to the first serotype. The ELISA, which has similar sensitivity to the serum neutralisation test, could be used as a rapid, easily performed test to identify fowl adenovirus-specific antibodies.     

Serotype IX, a Proposed New Streptococcus agalactiae Serotype

We identified three isolates of Streptococcus agalactiae (group B streptococcus [GBS]), of human origin, which failed to react with antisera against any of the nine known GBS serotypes. Polyclonal rabbit antisera raised against these isolates and standard GBS typing sera were used in capillary precipitation and Ouchterlony tests to compare the strains with known GBS serotype reference strains. All three previously nontypeable isolates reacted with all three new antisera, producing lines of identity in the Ouchterlony test. Weak cross-reactions with antisera against several GBS serotypes were observed but were removed by absorption with corresponding antigens. The new antisera were used to test 227 GBS isolates that had been nontypeable or difficult to type using standard antisera. Of these, five reacted with the new antisera. These results suggested that all eight isolates belong to the previously unrecognized GBS serotype. They were tested by Western blotting for the Calpha and Cbeta proteins and by PCR to identify molecular serotypes and surface protein antigen genes. Two segments of the cps gene cluster (3' end of cpsE-cpsF and 5' end of cpsG, approximately 700 bp; 3' end of cpsH and 5' end of cpsM, approximately 560 bp) were sequenced. All eight isolates expressed Calpha, and seven expressing the Cbeta protein and the corresponding genes, bca and bac, respectively, were identified. They all share the same, unique partial cps sequence. These results indicate that these eight isolates represent a new S. agalactiae serotype, which we propose should be designated serotype IX.     

Immunodiffusion test for serodiagnosing subcutaneous zygomycosis.

Culture filtrate antigens of Basidiobolus ranarum and Conidiobolus coronatus were analyzed by immunodiffusion (ID) with homologous rabbit antisera. B. ranarum and C. coronatus were each found to have five specific antigens. Results of tests with heterologous antisera indicated that all of the species shared at least one antigen. ID tests incorporating the specific precipitin bands as references were developed for detection of basidiobolomycosis and conidiobolomycosis. These tests were performed with sera from humans and horses with proven basidiobolomycosis and conidiobolomycosis as well as with control sera from humans and animals with and without heterologous mycotic and oomycotic infections. Only sera from cases of basidiobolomycosis and conidiobolomycosis produced lines of identity with the reference precipitates of B. ranarum and C. coronatus, respectively. The ID tests were found to be completely sensitive and specific for determining the etiology of zygomycosis caused by these two species. In addition they appeared useful for monitoring resolution of the infections.     

Improved O-serotyping method for Serratia marcescens.

In a previous study, we found that some O serotypes of Serratia marcescens, as defined by agglutination tests, were not based on lipopolysaccharide (LPS) O antigens. We developed a dot enzyme immunoassay with a high degree of LPS specificity and tested 104 distinct clinical strains. Only 7 of the 24 existing O antigens were found in more than one strain: O12/O14 (30.8% of strains examined), O21 (12.5%), O8 (8.7%), O6/O7 (5.8%), O4 (3.8%), O18 (2.9%), and O9 (2.9%). Two new antigens, S1254 (13.5%) and S3255 (3.8%), were also found. Agglutination tests with O antisera identified the LPS antigen in only 36 strains. Prodigiosin production was restricted to serotypes O8, O6, and S3255 and strains with a rough or semirough LPS phenotype. Dot immunoassay appears to offer greater accuracy than agglutination tests for serotype identification in S. marcescens.     

Serological studies and isolations of serotype hardjo and Leptospira biflexa strains from horses of Argentina.

Three pathogenic leptosipras and 12 saprophytic Leptospira biflexa strains were isolated from 72 apparently normal horse kidneys collected at an abattoir in Argentina. Cross-agglutination reaction patterns of the pathogens showed that they were antigenically homologous with members of the Hebdomadis group. When one of the strains was compared to Hebdomadis serotypes in reciprocal agglutination-absorption tests, it was found to be serologically homologous to serotype hardjo. This is the first known report of an isolation of this serotype from horses. Serological tests were also carried out on randomly collected abattoir sera from 245 horses to determine the prevalence of equine leptospirosis. Significant antibody titers (1:100 or greater) were found in 74.6% of the sera. Predominant reactions occurred with the antigens pomona, hebdomadis group, pyrogenes, tarassovi, and canicola. Agglutination tests performed with antigen prepared with one of the saprophytic biflexa isolates showed seropositive reactions in 99.1% of the equine sera, with agglutination titers ranging from 1:100 to 1:3,200. Absorption of selected horse sera with the saprophytic strain removed the agglutinins to Leptospira interrogans serotypes. This suggests the possibility that L. biflexa strains may act as an antigenic stimulus and account for some of the persistent multiple cross-reaction patterns of equine sera with pathogenic serotypes.     

Antigenic relatedness of glucosyltransferase enzymes from streptococcus mutans.

The antigenic relationship of glucosyltransferases (GTF) produced by different serotypes of Streptococcus mutans was studied by using a functional inhibition assay. Rat, rabbit, or hamster immune fluids, directed to cell-associated or supernatant-derived GTF, were tested against ammonium sulfate-precipitated culture supernatants containing GTF from seven strains of S. mutans representing six different serotypes. An antigenic relationship was shown to exist among GTF from serotypes a, d, and g, since both rat and rabbit antisera directed to serotype a or g GTF inhibited GTF of serotypes d and g similarly and both antisera also inhibited serotype a GTF. Furthermore, serum inhibition patterns indicated that GTF of serotypes c and e, and possibly b, are antigenically related to each other, but are antigenically distinct from GTF of serotype a, d, or g. Serum antibody directed to antigens other than enzyme (e.g., serotype-specific antigen or teichoic acid) had little effect on the inhibition assay. Salivas from rats immunized with cell-associated or supernatant-derived GTF exhibited low but consistent inhibition of GTF activity, which generally corresponded to the serum patterns. The sera of two groups of hamsters immunized with GTF (serotype g), enriched either in water-insoluble or water-soluble glucan synthetic activity, gave patterns of inhibition quite similar to those seen with sera from more heterogenous cell-associated or crude supernatant-derived GTF preparations. Both groups of hamster sera also gave virtually identical patterns, suggesting that the two enzyme forms used as antigen share common antigenic determinants. The results from the three animal models suggest that among the cariogenic organisms tested, two (serotypes a, d, g and b, c, e), or perhaps three (serotypes a, d, g; b; and c, e), different subsets of GTF exist that have distinct antigenic determinants within a subset.     

Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae serotype 5.

Serotyping of Haemophilus pleuropneumoniae and serologic assays for detection of serotype-specific antibody are problematic due to the potential cross-reactivity of the crude antigens used for raising immune serum or for serology. The capsular polymer (CP) of H. pleuropneumoniae serotype 5 was investigated for serotype-specific activity with antiserum to whole cells or with antiserum made monospecific to CP by adsorption with a capsule-deficient mutant. When antiserum to whole cells or monospecific antiserum to CP was tested against purified CP from serotypes 1 to 7 by immunodiffusion or enzyme-linked immunosorbent assay, only capsules of serotype 5 were reactive. In addition, only encapsulated serotype 5 cells reacted with serum monospecific to CP in an indirect immunofluorescent-antibody assay. Serotype-specific antibody was completely inhibited in each assay by preincubation of purified CP with the serum. Antiserum to whole cells of H. pleuropneumoniae serotype 5 contained antibodies to proteins and lipopolysaccharide; these antibodies cross-reacted with antigens of heterologous serotypes by dot-blot enzyme-linked immunosorbent assay and immunoblotting. The antigenic activity of CP was stable after heating for at least 30 min at 100 degrees C. High titers of antibody to CP were present in the sera of rabbits immunized intravenously with whole log-phase cells or in the convalescent sera of pigs experimentally infected with H. pleuropneumoniae serotype 5. However, the purified CP was poorly immunogenic in rabbits and swine. Our results indicate that the capsule is the serotype-specific antigen of H. pleuropneumoniae and that a monospecific antiserum to capsule or purified capsule should be used for serotyping or serologic assays, respectively.     

Antigenic diversity of the serotype antigen complex of Neisseria gonorrhoeae: analysis by an indirect enzyme-linked immunoassay

An indirect enzyme-linked immunoassay (ELISA) has been developed to analyze the antigenic profile of the outer membrane serotype complex of Neisseria gonorrhoeae. Antisera raised in rabbits to serotype-specific vesicles (SSV) reacted primarily with homologous SSV; however, there was significant cross-reactivity (less than 50%) with heterologous SSV. N. meningitidis SSV cross-reacted with all antigonococcal SSV but at a lower degree (less than 20%). Preimmune sera did not cross-react significantly with all antigonoccoccal SSV. The sensitivity of the ELISA was enhanced when the integral SSV proteins 1a and 2 were used as adsorbed antigen. Heterologous anti-SSV cross-reacted slightly, having ELISA values less than 15% of the homologous reaction. Antisera prepared by immunoabsorbent affinity columns were highly specific. Homologous affinity anti-SSV reacted only with proteins 1a and 2. The reaction of immune sera was inhibited by homologous proteins 1a and 2; lipopolysaccharide and proteins 1a and 2 isolated from heterologous serotypes did not inhibit the reaction. The reaction of affinity-purified antisera could be inhibited only by homologous protein 1a. By the use of affinity-purified antisera, a specific and highly sensitive ELISA was developed to analyze the antigenic profile of strains of N. gonorrhoeae.     

Evaluation of commercial antisera for serotyping heat-labile antigens of Campylobacter jejuni and Campylobacter coli.

Commercial antisera for serotyping 22 heat-labile antigens of Campylobacter jejuni and Campylobacter coli were evaluated by using 66 isolates from human and nonhuman sources. Test results were compared with results of tests using antisera produced at the Centers for Disease Control (CDC), Atlanta, Ga. All strains (three isolates of each of the 22 serotypes) were typeable with the CDC antisera. Of 66 test strains, 39 (59%) were typed as the same serotype with both sets of antisera. Twenty-four strains (36%), including two heat-labile serotype reference strains, were nonreactive with the commercial antisera, and three strains (4.5%) were typed as serotypes different from those obtained with CDC antisera. Five of the 22 commercial antisera correctly serotyped all homologous strains. Our study indicated that two polyvalent antiserum pools, 7 unabsorbed antisera, and 16 absorbed monovalent antisera are weak and need modification to enhance their antibody titers. Further studies are necessary to explain the antigenic change to a different serotype in three strains.     

Evaluation of slide agglutination and ring precipitation tests for capsular serotyping of Haemophilus pleuropneumoniae.

Rapid slide agglutination (RSA), quantitative plate agglutination, slow tube agglutination (STA), and ring precipitation (RP) tests were performed on 200 isolates of Haemophilus pleuropneumoniae by using the type sera produced in rabbits against five known serotype strains and one strain 202. RSA and RP tests both yielded the same results as those by STA. None of the agglutination procedures could be used for serotyping isolates that autoagglutinated in saline. The RP test was successfully used for serotyping such strains. The specificity of the RSA and RP tests was confirmed by cross-absorption studies. All of the isolates except two had strong serotype-specific activities. The most common serotype isolated in Quebec was serotype 1, followed by serotypes 5 and 2. None of the isolates belonged to serotypes 3 and 4. Only two isolates were found to be untypable; they could possibly belong to serotype(s) not yet defined. The RSA and RP tests may be at least as reliable as the STA test, but easier to perform, less expensive, and much more rapid than any of the other methods reported. Of all the procedures studied by us, the RP test proved to be the method of choice for serotyping H. pleuropneumoniae; hence, it should replace the STA test for serotyping H. pleuropneumoniae.     

Identification of Salmonella O antigens by coagglutination

This study concerns the preparation of reagents for identifying the somatic O antigens of Salmonella enteritidis. Coagglutination reagents (COAGs) with antibody fixed to killed and stabilized protein A-bearing staphylococci were prepared with antisera which were used for identifying the somatic O antigens of S. enteritidis by the slide agglutination test. The reactions of the COAGs were compared with those obtained with the grouping antisera in routine slide agglutination tests in which 41 or more serologically different Salmonella strains, representing most of the known groups, were used. One-third of the COAGs gave identical reactions to those of the slide agglutination antisera. The reactions of the other COAGs varied from the slide agglutination antisera results, some by many reactions and others by only a few. The coagglutination procedure was more reactive than the routine slide agglutination test and resulted in cross-reactions which were not observed in the original grouping antisera. More COAGs were specific when they were tested with alcohol-treated cultures than with live cultures. Coagglutination conserves antiserum, allowing about 12 times as many tests for a given volume of group-specific glycerolized antiserum as does the slide agglutination method.