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.     

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.     

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.     

O-antigen specificities of the serotype strains of Serratia marcescens.

O antigens of the 24 O-serotype strains of Serratia marcescens were investigated in dot enzyme immunoassay with whole-cell antigens and by immunoblotting with lipopolysaccharide (LPS) antigens. Three pairs of strains, O2/O3, O6/O7, and O12/O14, had indistinguishable LPS antigens, despite having distinct specificities in agglutination tests with whole-cell antigens. Strong cross-reactions were also found in LPS antigens from strains O9/O15, O17/O19, O10/O22, and O16/O20. No high-molecular-weight LPS corresponding to O-side-chain material was detected in strain O11 or O13. A panel of absorbed antisera was prepared to facilitate the detection of a reduced set of LPS antigens in a dot enzyme immunoassay. We conclude that there are discrepancies between the existing serotypes as defined by agglutination tests and the antigenic composition of LPS antigens extracted from the serotype strains and that surface antigens other than LPS make a major contribution to the definition of serotype in the species.     

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.     

Contribution to the differentiation of haemophilus-strains isolated from chickens. II: Serological examinations using the plate-agglutination test

A total of 31 field isolates, 23 of them belonging to the H. paragallinarum species were examined serologically by means of the rapid slide agglutination test (RSA-test). One H. paragallinarum strain of serotype A, one of serotype B, and one serologically unclassified strain from California were used as reference strains. In addition, one strain of H. parainfluenzae was 'included 'in these studies. On the basis of the results of the RSA-tests With rabbit antisera 2 distinct serotypes of H. paragallinarum were differentiated. Cross reactions were observed among the serotypes which could be eliminated by dilution and absorption of the antisera. Eight of 23 H. paragallinarum isolates belonged to serotype A and 13 to serotype B. Two isolates showed spontaneous agglutination in 0.85% saline. Seven of 8 other isolates of Haemophilus bacteria, representing a culturally and biochemically separate group, could be distinguished from H. paragallinarum strains without difficulty. One strain showed spontaneous agglutination. The other 7 isolates formed 4 distinct serotypes on the basis of the RSA-tests, of which 3 isolates belonged to serotype 1, 2 to serotype 2, and one each to serotypes 3 and 4. Weak cross reactions were observed among these serotypes. No strains isolated from chickens included in this study were serologically identical with the strain of H. parainfluenzae.     

Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae is the causative agent of swine dysentery, a contagious mucohemorrhagic disease of the colon. Diagnosis of swine dysentery is extremely difficult because of the presence of cross-reactive antibodies to the proteins of S. hyodysenteriae and Serpulina innocens, a nonpathogenic inhabitant of the porcine large intestine. Therefore, monoclonal antibodies (MAbs) against the serotype-specific lipooligosaccharide (LOS) antigens of S. hyodysenteriae were produced to rapidly differentiate S. hyodysenteriae from S. innocens. Whole-cell preparations of S. hyodysenteriae serotypes 1 through 7 were used as antigens. MAbs were characterized by an indirect enzyme-linked immunosorbent assay with whole-cell or LOS antigen and by Western blot (immunoblot) analysis with whole-cell lysates as antigen. A total of 12 LOS-specific MAbs which could identify and differentiate the seven original serotypes of S. hyodysenteriae were produced. The MAb serospecificities are as follows: MAb 9G8, serotype 1; MAb 31D9, serotype 2; MAb 7D3, serotypes 2 and 7; MAb 24B7, serotype 3; MAb 13C2, serotype 4; MAb 18E9, serotype 4; MAb 2B7, serotype 6; MAb 1D2, serotypes 2, 5, and 7; MAb 9C5, serotypes 2, 5, and 7; MAb 11C9, serotype 7; MAb 11E10, serotype 7; and MAb 6G11, serotype 7.     

Serotyping of Pasteurella multocida isolated from swine lungs collected at slaughter.

We serotyped 222 Pasteurella multocida strains isolated from swine lungs at slaughter. Capsular serotypes A and D were determined by the hyaluronidase sensitivity and acriflavin agglutination tests, respectively. Somatic antigens were determined by gel diffusion against standard antisera. Capsular serotype A was found in 97.3% of the strains and serotype D in the remaining 2.7%. The primary somatic antigen most commonly found was type 3 (86.0%). Type 5 was also very common (88.7%), but was usually a secondary antigen. The most common overall serotype was A:3(5) (39.2%). Other common serotypes were: A:3(4,5,12) (12.2%); A:3(4,5) (11.2%); A:3(5,12) (10.4%); A:3 (6.8%); and A:5 (6.8%). Type D strains had a similar distribution of serotypes, but with a higher prevalence of D:5 (33.3%) and D:3(5) (33.3%).     

Comparison of some Shigella flexneri (Escherichia coli) hybrids in cross agglutination and agglutinin absorption tests.

Antigenic structure of Lac+ hybrids selected from Escherichia coli x Shigella flexneri crosses was compared in the cross agglutination and absorption tests. Identity of the hybrids from the recipients of serotype 2a and variant Y was proved, as well as from serotypes 1b and 4b. The latter hybrids were coupled with Shigella flexneri strains 3a, 3b and 3c. The hybrids appeared almost identical with serotype 3c.     

Yersinia enterocolitica isolates from humans in California, 1968-1975.

This paper reports on the serological and biochemical characteristics of 24 human isolates of Yersinia enterocolitica submitted to the California Department of Health from 1968 through 1975. Nine different serotypes were represented. The majority of strains were serotype O:8 (six strains) and serotype O:5 (five strains). Sources of the isolates included feces (12 cases), blood (3), sputum or throat (3), bile or bowel drainage (2), wounds (2), breast abscess (1), and skin abscess (1). Clinical histories indicated a number of different syndromes. Underlying medical conditions existed in 13 cases. Results of selected biochemical tests and antimicrobial susceptibility tests on the strains indicated grouping compatible with the O serotypes of the organisms.     

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.     

The cps Genes of Streptococcus suis Serotypes 1, 2, and 9: Development of Rapid Serotype-Specific PCR Assays

We developed three type-specific PCR assays for the rapid and sensitive detection of Streptococcus suis serotype 1 (plus 14), serotype 2 (plus 1/2), and serotype 9 strains in tonsillar specimens from pigs. The PCR primers were based on the sequences of type-specific capsular genes of S. suis serotype 1, 2, and 9 strains. We recently characterized a major part of the capsular biosynthesis (cps) locus of S. suis serotype 2. Here we extended these studies and characterized major parts of the cps loci of S. suis serotypes 1 and 9. Type-specific genes were identified by cross-hybridization experiments between the individual cps genes and chromosomal DNAs from the 35 different serotypes. Four genes of S. suis serotype 1 specifically hybridized with serotype 1 and 14 strains only. Five genes of S. suis serotype 2 specifically hybridized with serotype 2 and 1/2 strains only, and two genes of S. suis serotype 9 specifically hybridized with serotype 9 strains. Until now rapid and sensitive diagnostic tests were available only for pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1. The serotype-specific PCR assays can therefore be useful tools for the identification of serotype 1, 14, 2, 1/2, and 9 strains both for diagnostic purposes and in epidemiological and transmission studies. Therefore, these tests may facilitate control and eradication programs.     

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.     

Serotypes of 'Pasteurella' anatipestifer isolates from ducks in Singapore: a proposal of new serotypes

'Pasteurella' anatipestifer (Pa) isolates from local ducks were typed by slide and tube agglutination tests using antisera against representative strains of existing serotypes. As the strains of serotype 13 and serotype 17 were found to be serologically identical, it is proposed that they be jointly designated as serotype 13. It is also proposed that the English serotype P, represented by strain HPRS 2565, be adopted as a replacement for the existing serotype 4 which has been excluded as Pa. Three new serotypes were identified among the 352 isolates from ducks in Singapore. It is proposed that these new serotypes be designated as serotypes 17, 18 and 19 under the present classification system.     

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes.

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec.     

Serotypic classification of hantaviruses by indirect immunofluorescent antibody and plaque reduction neutralization tests

Antisera prepared against 16 strains of hantaviruses isolated from patients with hemorrhagic fever with renal syndrome (HFRS) or from rodents captured in HFRS-endemic and nonendemic regions were titrated against Hantaan virus strain 76-118, Puumala virus strain Sotkamo, and Prospect Hill virus strain Prospect Hill-I by using the indirect immunofluorescent antibody and plaque reduction neutralization tests. Isolates fell into one of four distinct groups or serotypes. Serotype 1 included Apodemus-derived strains, serotype 2 included Rattus-derived strains, serotype 3 included Clethrionomys-derived strains, and serotype 4 included Microtus-derived strains. Serotypic classification of hantavirus infections was possible for humans and rodents in widely varied geographical areas, but in a few instances, sera from patients with HFRS did not conform to any of the four serotypes, suggesting the existence of as yet unidentified serotypes. A definitive serological classification of hantaviruses must await analysis of additional virus isolates.     

Development of monoclonal antibodies against Ureaplasma urealyticum serotypes and their use for serotyping clinical isolates

Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.     

Some antigenic properties of Haemophilus parasuis and a proposal for serological classification

We propose a serological classification of Haemophilus parasuis into at least five serovars, using an agar-gel-precipitation test with extracts from autoclaved cells. Thirty-two strains were examined, and it was possible to classify 26 of them. The specific antigens were thermostable and soluble and were not affected by pronase treatment but could be extracted by phenol, suggesting a polysaccharide. This polysaccharide seemed to be identical with the capsular substance of serovars 1, 2, and 3. In the presumably uncapsulated strains of serovars 4 and 5, the specific antigen was probably located in the outer membrane. The diversity of the specific substance within the different serotypes was shown by the differences in their electrophoretic migration patterns. Other extraction procedures showed that the washing supernatant and extracts at 60 and 100 degrees C were identical with the 121 degrees C extracts for serovars 1, 2, and 3. In serovars 4 and 5, washing antigens, if present, were different from 100 and 121 degrees C extracts. Other common antigens, presumably proteinaceous antigens, were detected after extraction at 60 and 100 degrees C. The slide and tube agglutination tests allowed classification only for the capsulated strains of serovars 1, 2, and 3. The specific agglutinogens were very sensitive to incubation temperature, and the absorption test showed them to be identical with the 121 degrees C precipitinogens.     

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.     

Production and characterization of agglutinating monoclonal antibodies against predominant antigenic factors for Candida albicans.

Two clones, CA4-2 and CA5-4, which produced agglutinating monoclonal immunoglobulin M (IgM) antibodies (MAbs) against mannan antigens of Candida albicans serotype A, were established. The specificity of each MAb was determined by slide agglutination tests for cross-reactivity patterns against the homologous and six other strains of Candida and a strain of Torulopsis: C. albicans serotype B, C. tropicalis, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, and Torulopsis glabrata. The MAb produced by CA4-2 reacted with the homologous, C. tropicalis, and T. glabrata strains, whereas the MAb produced by CA5-4 reacted with the homologous, C. albicans serotype B, and C. tropicalis strains. These results are consistent with results obtained by comparative experiments with several strains of each serotype or species. Specificity of these two MAbs by agglutination was also consistent with the cross-reactivity patterns demonstrated by indirect immunofluorescence staining. The competitive binding experiments by immunofluorescence staining with two MAbs and polyclonal factor sera (PAb factors) 5 and 6 suggested that the MAb from clone CA4-2 did not completely correspond to PAb factor 6 and that the MAb from CA5-4 was distinct from PAb factor 5 in its manner of binding to determinants (the latter was designated 5b), Cross-reactivity patterns, however, furnished evidence that these two MAbs could replace the known PAb factors 6 and 5, respectively, as reagents for aid in the identification of the strains of C. albicans and their serotypes.