Serovar-specific Antigens Research Articles

Identification of the Chromosomal Region Essential for Serovar-Specific Antigen and Virulence of Serovar 1 and 2 Strains of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae causes swine erysipelas, an infection characterized by acute septicemia or chronic endocarditis and polyarthritis. Among 17 E. rhusiopathiae serovars, determined based on heat-stable peptidoglycan antigens, serovars 1 and 2 are most commonly associated with the disease; however, the molecular basis for the association between these serovars and virulence is unknown. To search for the genetic region defining serovar 1a (Fujisawa) strain antigenicity, we examined the 15-kb chromosomal region encompassing a putative pathway for polysaccharide biosynthesis, which was previously identified in the E. rhusiopathiae Fujisawa strain. Six transposon mutants of Fujisawa strain possessing a mutation in this region lost antigenic reactivity with serovar 1a-specific rabbit serum. Sequence analysis of this region in wild-type strains of serovars 1a, 1b, and 2 and serovar N, which lacks serovar-specific antigens, revealed that gene organization was similar among the strains and that serovar 2 strains showed variation. Serovar N strains displayed the same gene organization as the serovar 1a, 1b, or 2 strain and possessed certain mutations in this region. In two of the analyzed serovar N strains, restoration of the mutations via complementation with sequences derived from serovar 1a and 2 strains recovered antigenic reactivity with 1a- and 2-specific rabbit serum, respectively. Several gene mutations in this region resulted in altered capsule expression and attenuation of virulence in mice. These results indicate a functional connection between the biosynthetic pathways for the capsular polysaccharide and peptidoglycan antigens used for serotyping, which may explain variation in virulence among strains of different serovars.

Seroepidemiology of canine leptospirosis by iELISA and MAT

Aim: The present study was conducted to determine the prevalence of antibodies against leptospira using a genus specific and serovar specific antigen by indirect ELISA (iELISA) and Microscopic Agglutination test (MAT), respectively. Materials and Methods: 300 sera samples were collected from dogs, from Pondicherry, suspected to be suffering from Canine leptospirosis and with no history of vaccination. Using the outer membrane proteins (OMP's) of Leptospira interrogans serovar canicola as antigen the leptospiral antibodies were detected by iELISA and compared with the MAT using five commonly infecting serovars (canicola, icterohaemorrhaegiae, grippotyphosa, pomona, australis) as antigens. Statistical analysis of the data was carried out using chi-square test. Result: Out of 300 sera samples, 159 (53 %) and 99 (33 %) sera samples were found to be positive for leptospiral antibodies by iELISA and MAT, respectively. The iELISA employed in this study revealed a very high sensitivity of 94.94 % in comparison with the MAT (59.11 %). However, iELISA showed less specificity (67.66 %) when compared to MAT (96.45 %). By chisquare test, a significant difference (P<0.001) was obtained between the results from iELISA and MAT. Conclusion: The present study revealed that the iELISA was found to be highly sensitive, rapid and easy to perform in comparison with MAT for the detection of canine anti-leptospiral antibodies.

Down-Regulation of Key Virulence Factors Makes the Salmonella enterica Serovar Typhimurium rfaH Mutant a Promising Live-Attenuated Vaccine Candidate

Mutants of Salmonella enterica serovar Typhimurium that lack the transcriptional regulator RfaH are efficient as live oral vaccines against salmonellosis in mice. We show that the attenuation of the vaccine candidate strain is associated with reduced net growth in epithelial and macrophage cells. In order to identify the relevant RfaH-dependent genes, the RfaH regulon was determined with S. enterica serovars Enteritidis and Typhimurium using whole-genome Salmonella microarrays. As well as impacting the expression of genes involved in lipopolysaccharide (LPS) core and O-antigen synthesis, the loss of RfaH results in a marked down-regulation of SPI-4 genes, the flagellum/chemotaxis system, and type III secretion system 1. However, a proportion of these effects could have been the indirect consequence of the altered expression of genes required for LPS biosynthesis. Direct and indirect effects of the rfaH mutation were dissociated by genome-wide transcriptional profiling of a structural deep-rough LPS mutant (waaG). We show that truncation of LPS itself is responsible for the decreased intracellular yield observed for DeltarfaH strains. LPS mutants do not differ in replication ability; rather, they show increased susceptibility to antimicrobial peptides in the intracellular milieu. On the other hand, evidence that deletion of rfaH, as well as some other genes involved in LPS biosynthesis, results in enhanced invasion of various mammalian cells is shown. Exposure of common minor antigens in the absence of serovar-specific antigens might be responsible for the observed cross-reactive nature of the elicited immune response upon vaccination. Increased invasiveness of the Salmonella rfaH mutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders the rfaH mutant an ideal vaccine candidate.

Geographic distribution of Mycobacterium avium-intracellulare complex serovars isolated from patients in five cities of Japan

Mycobacterium avium-intracellulare complex (MAC), isolated from patients with MAC infection in five cities in Japan, was classified into M. avium and M. intracellulare by DNA-DNA hybridization assay (DDH). They were also classified into serovars by seroagglutination test and thin-layer chromatography (TLC) detecting serovar-specific antigens. The ratios of M. avium: intracellulare isolated in Sendai, Maebashi, Kobe, Hamamatsu, and Kumamoto, were 100:0, 26:16, 37:12, 37:39, and 17:54, respectively. It was high in the northern city, Sendai, whereas the ratio in the southern city, Kumamoto, was low. The main serovars detected in Sendai and Maebashi were 8 and 4. Various serovars including a new type of M. intracellulare were detected with main serovars 16 and 7 in Kumamoto. M. intracellulare including main serovar 16, and M. avium including main serovar 4, were detected in the same ratio in Hamamatsu. M. avium including main serovars 8 and 4, was detected in considerable high ratio in Kobe. Geographic distribution was observed in both species of MAC and serovars.

Recognition of Leptospira interrogans antigens by vaccinated or infected dogs

Antigenic recognition of leptospiral antigens by vaccinated or infected dogs was studied by microagglutination test (MAT) and by western blots. In western blots, serovar specific antigens detected by MAT migrated in the 18–31 kDa zone. The 25–31 zone seemed to be linked to antigens indicating virulence of the strain. These antigens are LPS. The first antibodies made after infection are produced against LPS migrating in the 14 kDa zone. Many protein antigens are common in leptospires belonging to different serogroups. Virulent strains exhibited specific antigens in the 45 and 32–34 kDa zones.

Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis.

Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38-40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38-40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.

Protective activity of glycolipid antigen against infection by Leptospira interrogans serovar canicola

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23-30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.

Specificity of Salmonella Porin as an Eliciting Antigen for Cell‐Mediated Immunity (CMI) Reaction in Murine Salmonellosis

The specificities of Salmonella porin on elicitations of delayed-type hypersensitivity (DTH) reaction and interleukin-2 (IL-2) production in BALB/c mice immunized with Salmonella typhimurium were examined. Only porin from S. typhimurium was capable of eliciting significant levels of DTH and IL-2 production in S. typhimurium-immunized mice, whereas no significant DTH and IL-2 production were induced by porin from Salmonella enteritidis or Escherichia coli. Our observations suggested that Salmonella porin was a serovar-specific antigen for the elicitation of cell-mediated immunity (CMI) in salmonellosis.

Can group- and serovar-specific proteins be detected in Ureaplasma urealyticum?

Ureaplasma urealyticum has been subspeciated by a number of serologic methods. Eight serotypes have been identified by modified metabolic inhibition, growth inhibition and indirect hemagglutination. Fourteen serovars have been identified by immunofluorescence and 16 by the mycoplasmacidal assay. The present studies were performed to determine if group-specific antigens could be detected by immunofluorescence and if group- or serovar-specific antigens could be detected by enzyme-linked immunosorbent assay and immunoblotting. Reaction of rabbit antisera to U. urealyticum with homologous and heterologous serotypes based upon end point immunofluorescent titration did not differentiate the serovars into the two biotypes. In attempts to quantitate the number of a given serovar present in clinical specimens, the total number of colonies that were stained often exceeded 100%, suggesting either that the serovars represented in stock cultures are not truly representative of those present in humans or that certain strains express multiple serovar specificities. End point titration in an enzyme-linked immunoadsorbent assay using either whole cells or cell lysates and rabbit antisera failed to distinguish between group and serovars (i.e. in many cases end point titers were only 2-fold lower with heterologous antigens). Furthermore convalescent sera from patients infected with a single serovar failed to induce a serovar-specific response detectable in an enzyme-linked immunosorbent assay. Likewise immunoblotting of one dimensional electrophoretograms with sera from humans known to be infected with a single serovar showed that antibodies against that serovar recognized peptides present in all serovars and reacted with those bands with various degrees of intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination, toxigenicity and sorbitol fermentation of Clostridium difficile.

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic-associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol-treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross-agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120-10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar-specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic-associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that sorbitol-fermenting property of C. difficile if very closely related to the toxigenicity and agglutinability.