Comparison of the Complement-Fixation Test and the Microscopic-Agglutination Test (Agglutination-Lysis) for the Detection of Leptospiral Serogroup Antibodies

The MA and CF tests using alcohol extracted antigens have been compared with sera from infected rabbits and calves. The MA test was highly serotype specific with serum from both animal species. The CF test was broadly reactive with serum from rabbits infected with various serotypes. However, when bovine serum was used cross reactivity was reduced and it was necessary to use pooled antigens to detect heterologous serotypes.     

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.     

Wall-associated protein antigens of Streptococcus mutans.

When heat-killed whole organisms of Streptococcus mutans strain Ingbritt (serotype c) were injected into rabbits, antibodies to at least 12 antigens were detectable by crossed immunoelectrophoresis. In contrast, when rabbits were immunized with organisms which had been subjected to extraction with the detergent sodium dodecyl sulphate (SDS), antibodies to only two protein antigens were found. These two proteins (A and B), while existing in a form apparently closely associated with peptidoglycan, could also be recovered from homogenates of whole organisms after sonication and from culture filtrates. Antigenic material was excreted throughout growth. SDS-polyacrylamide gradient gel electrophoresis showed A to have a molecular weight of 29 000, while B had a molecular weight of 190 000. Antigen B was purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. All of six strains of serotype c examined produced antigen B. Strains of serotypes e and f also produce antigenically identical proteins and strains of serotypes d and g produce proteins which cross-reacted with antigen B. Antigen B was specifically precipitated by rabbit antiserum to human heart tissue.     

Solubilization of antigens of Fasciola hepatica which react with antibodies to Schistosoma mansoni.

Extracts of Fasciola hepatica adult worms contain antigens reactive with antisera prepared against Schistosoma mansoni adult worms. These antigens are poorly solubilized when homogenized in a phosphate buffered saline (PBS) solution and pellet readily when subjected to high speed centrifugation. Solubilization is improved greatly by the addition of sodium dodecyl sulfate (0.03%) to the PBS. When this is done, one obtains approximately the same total amount of crude Lowry reactive material as with PBS extraction followed by high speed centrifugation but antigenic reactivity to an anti-S. mansoni antiserum increases enormously. The antigens liberated from F. hepatica SDS extraction are largely materials under 200,000 MW and over 60,000 MW.     

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae

Abstract Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognize serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG_2a, IgG_2b, and IgG₃ were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.     

Immunochemical characterization of Neisseria meningitidis serotype antigens by immunodiffusion and SDS-polyacrylamide gel electrophoresis immunoperoxidase techniques and the distribution of serotypes among cases and carriers

The chemical nature of the antigens of the meningococcal serotypes described by Frasch and colleagues was determined by a combination of immunodiffusion and the SDS-polyacrylamide gel electrophoresis immunoperoxidase technique (SGIP). It was confirmed that the serotype antigens of the outer membrane of serotypes 1, 2, 6, 9, 11 and 12 were proteins, whilst those of serotypes 4,5 and 8 were lipopolysaccharides. Serotype 2 can now be divided into three related types, provisionally called 2a (originally serotype 2), 2b and 2c with the specific antigens being proteins having molecular weights of 41,000, 41,500 and 41,500, respectively. A total of 195 strains of meningococci isolated from patients and carriers in the Netherlands and 20 serogroup Y strains from patients in the U.S.A. were serotyped by means of immunodiffusion. Serotype 2a could be demonstrated in some strains belonging to the serogroups B (only those from carriers), C, W-135 and Y (only those from the U.S.A.). The W-135 strains isolated from patients in this series more often belonged to serotype 2a than did the W-135 strains from carriers. Serotype 2b was present in about half of the serogroup B and a few serogroup C strains isolated from patients with meningitis, but absent in serogroup B and C strains from carriers. Serotype 2c could only be demonstrated in serogroup Y strains, both from the Netherlands and the U.S.A. The other serotypes were found only sporadically.     

Characterization and Relatedness of Marine Vibrios Pathogenic to Fish: Physiology, Serology, and Epidemiology

Cultural characteristics and serological relationships of pathogenic marine vibrios isolated from fish in the Pacific Northwest were studied. These organisms were compared with cultures of Vibrio anguillarum, a known fish pathogen. On the basis of morphological and cultural characteristics, the Pacific Northwest strains of Vibrio were found to be closely related to V. anguillarum. Serological analyses of thermostable antigens served to distinguish three serotypes among the vibrios. Serotype 1 was composed of organisms isolated from Northwest salmonids; serotype 2 of strains of V. anguillarum from European waters; and serotype 3 of organisms isolated from Pacific herring. The epidemiology of vibrio disease among populations of fish in the Pacific Northwest is discussed.     

Serological Characterization of Haemophilus Pleuropneumoniae (Actinobacillus Pleuropneumoniae) Strains and Proposal of a New Serotype: Serotype 9

Ten strains of H. pleuropneumoniae isolated from 10 herd outbreaks of pleuropneumonia were studied by means of the slide agglutination test, the indirect haemaggluitiniation (IHA) test and by gel diffusion. The strains were antigenically homogeneous and serologically distinct from serotypes 1 through 8. It is therefore proposed to refer these strains to a new serotype: serotype 9, with strain CVJ 13261 as the type strain. In addition to the serotype-specific capsular antigens, capsular antigen of serotype 1 (strain 4074) could be demonstrated in the 10 strains by means of gel diffusion analyses. In cross protection studies it was shown that the antigenic determinants shared by serotypes 9 and 1 were unable to yield a sufficient protection against disease. Thus, parenteral immunization with a killed 6-h culture of serotype 9 did not afford an acceptable protection against challenge with serotype 1 since only 3 of the 5 vaccinates were protected. The reverse experiment showed that parenteral immunization with serotype 1 only protected 1 out of 4 vaccinates.     

Enzyme-linked immunosorbent assay in the diagnosis of campylobacteriosis

Campylobacter jejuni and Campylobacter coli are the bacterial cause of human gastroenteritis commonly reported worldwide. The serodiagnosis of Campylobacter infections is not routinely done in Poland so the aim of this study was to evaluation of ELISA in the diagnosis ofcampylobacteriosis. Serum samples obtained from 145 patients with gastroenteritis were tested by ELISA with 7 different heat-stable antigens of C. jejuni and one of C. coli and by the commercial Virion/Serion ELISA with purified 45 kDa outer membrane protein of C. jejuni. Antibodies for heat-stable antigens of C. jejuni were detected statistically more often than antibodies for heat-stable antigens of C. coli and for purifled protein of C. jejuni. We found significant differences in the frequency of detection of antibodies to different heat-stable antigens, ranged from 18.6% to 68.9% of positive results, what indicate for serological heterogenicity of C. jejuni strains isolated in Poland. The results of our study showed usefulness of ELISA in serological diagnosis of campylobacteriosis. However it is necessary to serotype the C. jejuni strains isolated in Poland to find the appropriate C. jejuni serotype for using in ELISA.     

Muskrats as carriers of pathogenic leptospires in The Netherlands

Leptospires were isolated from 24 of 327 (7%) muskrats (Ondatra zibethicus) caught in The Netherlands. All isolates were identified asLeptospira interrogans. One isolate was typed as serovarcopenhageni in the Icterohaemorrhagiae serogroup, one as serovarlora in the Australis serogroup. Twenty-one isolates showed a close relationship with serovarsgrippotyphosa, valbuzzi, muelleri andratnapura from the Grippotyphosa serogroup. One isolate was lost. Sera from 196 muskrats were examined by the microscopic agglutination test. Forty-five (23%) sera reacted positively (titers1: 160), 42 (21%) of these 45 sera to Grippotyphosa and 3 (2%) to Sejroe serogroup antigens. This is the first report of serological and cultural evidence of leptospira infection in muskrats in The Netherlands.Abbreviations CAAT cross agglutination absorption test - 5-FU 5-fluorouracil - MAT microscopic agglutination test - MCA monoclonal antibodies - PBS phosphate buffered saline - REA restriction endonuclease analysis - SDS sodium dodecyl sulphate     

Determination and Analysis of Actinomyces israelii Serotypes by Fluorescent-Antibody Procedures

This study was undertaken to determine the antigenic relationships between serotypes of Actinomyces israelii with fluorescent-antibody (FA) procedures. In addition, the antigenic relationships between A. israelii and other members of the genus Actinomyces were studied by the same methods. Seven FA conjugates were used to determine the serological characteristics of 28 isolates believed to represent A. israelii, serotypes 1 and 2. The results showed that the lower dilutions of serotype 1 conjugates stained serotype 2 antigens; however, serotype 2 conjugates did not stain serotype 1 antigens. Serotype 1 conjugate could be made specific by adsorption. A. israelii serotype 1 conjugate cross-reacted also with A. naeslundii, but this cross-reaction could be eliminated by adsorption or dilution. Serotype 2 conjugate appeared to be specific for A. israelii serotype 2. Adsorption studies revealed antigenic variants among the various A. israelii serotype 1 and 2 isolates. However, all isolates could be identified by direct FA staining with appropriate conjugates. One isolate previously identified as A. israelii was shown, on the basis of FA studies, to be an A. naeslundii. A polyvalent diagnostic reagent was prepared which was specific for A. israelii serotypes 1 and 2. This reagent should find application in diagnostic and reference laboratories.     

Western blot (immunoblot) analysis of the fimbrial antigens of Bacteroides nodosus

The roles of the fimbrial subunit and the putative basal protein antigens in the serological classification of Bacteroides nodosus have been examined by Western blot (immunoblot)-antibody binding studies of fimbriae isolated from a wide range of strains representative of different serogroups and serotypes. Fimbrial subunits were recognized by antiserum against the homologous serogroup but not generally by heterologous antisera, whereas recognition of the basal antigen was independent of serological classification. Secondary cross-reaction patterns among fimbrial subunits indicated that some serogroups may be more closely related than others. Examples include serogroups C and G and serogroups D and H. Similar analyses of isolates classified within serotypes A1 and A2, with serotype-specific antisera, showed that this subdivision is also determined by the fimbrial subunit and that significant variation does occur even at this level. These studies suggest that the various serogroups and serotypes of B. nodosus comprise a series of overlapping sets of antigenically related strains.     

Virulence of Escherichia coli Serotypes for Mice

A study was undertaken to determine whether virulence in mice could be used to assess the pathogenicity of a variety of Escherichia coli serotypes. Sixty-one E. coli strains isolated from animals, poultry, or humans were serotyped to determine their O, K, and H antigens, and were administered to mice via the intraperitoneal route with and without a mucin adjuvant. The ld(50) dose was then determined for each serotype. The results indicated that the source of the serotype may be associated with virulence for mice. Serotypes isolated from nonenteric, systemic sources showed a greater virulence for mice inoculated intraperitoneally than did the enteric and the nonenteric, nonsystemic (localized) isolates. It was observed that not all serotypes belonging to a specific serogroup were virulent for mice and that the presence or absence of a K antigen had no effect on the virulence of strains of one serotype.     

Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3.

Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized. The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V. anguillarum serovars was also evaluated. The three serological assays used to establish the serogroups within V. anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected. Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera. Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V. anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns. On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V. anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay. From these results, it can be concluded that V. anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist.     

Immunochemical study of nutritionally variant streptococci

Nutritionally variant streptococci (NVS) have been characterized by their growth as satellite colonies around colonies of staphylococci or several other gram-positive or gram-negative bacterial strains. The majority of the NVS strains were isolated from patients with subacute bacterial endocarditis. Organisms identified as NVS were subdivided into three serotypes by rocket-line electrophoresis and hemagglutination inhibition assays. Ninety-nine of 103 strains expressed one or more of the three serotype antigens; however, a group antigen was not demonstrated in the various extracts of these streptococci. Surface protein studies confirmed the NVS differentiation into serotypes. Serotype I organisms expressed surface protein(s) specific for the serotype, whereas the serotype II and III NVS demonstrated common protein(s) on their surface. Furthermore, SDS extraction released a greater amount of radioiodinatable surface protein from serotypes I and III bacteria than serotype II. Finally, there was no correlation between the serotype or the disease of the patients from which the NVS strains were isolated.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. IV. Specificity of antigens

The specificity of the lipopolisacharydes and released proteins (Yop) of Yersinia was tested using the sera of rabbits immunised with pathogenic and non-pathogenic strain of Y. enterocolitica and Y. pseudotuberculosis as well as selected sera of patients. The results of this study showed a cross-reactions between the different serotypes of Y. enterocolitica with the strongest reactions between the pathogenic serotypes O:3 and O:9 and pathogenic serotype O:5,27 and non-pathogenic serotype O:5. Sera positive for B. burgdorferi and from patients with Graves' disease showed a slight cross-reactivity with Yop proteins of Yersinia. However, the higher cross-reactivity was observed between the LPS of Yersinia and Salmonella spp. Due to the evidence of cross-reactivity the results of serological investigations should be interpreted with caution.     

Isolation and characterisation of the phospholipase B gene of Cryptococcus neoformans var. gattii

Cryptococcus neoformans var. gattii (serotypes B and C) is a human pathogen, ecologically, biochemically, clinically and genetically different from C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). The phospholipase B (PLB1) gene from serotypes B and C was isolated and characterised. It resembled the serotype A and D genes, with an overall sequence homology of more than 85%. The respective open reading frames were 2236 bp (serotype B) and 2239 bp (serotype C) in length. Each contained six introns and encoded a 68-kDa protein destined for secretion. PLB1 was located on the second smallest chromosome in both serotypes. Gene expression, measured as mRNA, was not regulated by temperature, pH or exogenous nutrients.     

Predicting Salmonella enterica serotypes by repetitive sequence-based PCR

Repetitive extragenic palindromic sequence-based PCR (rep-PCR) utilizing a semi-automated system, was evaluated as a method to determine Salmonella serotypes. A group of 216 Salmonella isolates belonging to 13 frequently isolated serotypes and one rarer serotype from poultry were used to create a DNA fingerprint library with the DiversiLab System software. Subsequently, a blinded set of 44 poultry isolates were fingerprinted and queried against the library in an attempt to putatively assign a serotype designation to each Salmonella isolate. The query isolates were previously typed employing standard serological techniques. Utilizing pair-wise similarity percentages as calculated by the Pearson correlation coefficient, the predicted serotype of 28 isolates matched the serological typing result. For eight isolates, rep-PCR results were interpreted as one of two very closely-related serotypes, Hadar and the rarer Istanbul. Traditional serological assays have difficulty distinguishing between these groups, and sequencing interspacer regions of the rrfH gene was unable to differentiate among isolates of these two serovars. Six of the remaining isolates resulted in no match to the database (similarity values <95%) and these indeed proved to be serotypes not included in the original library. The two remaining samples proved discrepant at the 95% similarity threshold, however examination of electropherograms clearly indicated fingerprint variability between query and library samples, suggesting an expanded rep-PCR library will be necessary for increased utility. Since serological assays can take several days to weeks to provide information, the DiversiLab System holds promise for more rapid serotype classification for members of this group.     

A novel serotype-specific gene cassette (gltA-gltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b

Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall.     

A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites; no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.