Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species.

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.     

Evaluation of microagglutination test for differentiation between Serpulina (Treponema) hyodysenteriae and S. innocens and serotyping of S. hyodysenteriae.

Swine dysentery is a mucohemorrhagic diarrheal disease caused by the anaerobic spirochete Serpulina hyodysenteriae. At present, the serotyping is done by immunodiffusion testing with lipopolysaccharide (LPS) extract as antigen and rabbit hyperimmune sera produced against different serotypes of S. hyodysenteriae. Since the preparation of LPS is time-consuming and requires a large quantity of bacteria, it is desirable to use a serotyping method which does not require the extraction of LPS. In the present investigation, microagglutination was evaluated by using both formalinized whole- and boiled-cell suspensions as antigens and rabbit hyperimmune sera produced against formalinized whole-cell suspensions of reference strains of S. hyodysenteriae and S. innocens B256. Use of boiled cell suspension as antigen permitted the differentiation between isolates of S. hyodysenteriae and S. innocens as well as serotyping of S. hyodysenteriae strains accurately. A total of 18 isolates were identified as S. hyodysenteriae, and 3 isolates were identified as S. innocens. The microagglutination test was found specific, sensitive, and easy to perform; thus, it was judged suitable for routine identification and serotyping of S. hyodysenteriae isolates.     

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.

Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae.     

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.     

Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using total DNA obtained from normal swine feces inoculated with decreasing concentrations of S. hyodysenteriae cells, the sensitivity of the PCR assay was calculated to be between 1 and 10 organisms per 0.1 g of feces. The PCR assay was 1,000 times more sensitive than conventional culture of dysenteric feces on selective medium. There was complete agreement between the results of PCR assays and anaerobic culture on selective agar medium with diagnostic specimen (n = 9) obtained from six farms on which there were cases with clinical signs suggestive of swine dysentery. Detection of S. hyodysenteriae by PCR amplification of DNA has great potential for rapid identification of S. hyodysenteriae in diagnostic specimens.     

Characterization of two DNA probes specific for Serpulina hyodysenteriae.

Two DNA probes, one 1.1- and one 0.75-kb probe, specific for Serpulina hyodysenteriae were isolated from a genomic library generated from virulent S. hyodysenteriae 5380. These probes are highly specific and react with all S. hyodysenteriae strains tested. Under stringent conditions, the DNA probes did not react with the nonpathogenic species Serpulina innocens or with other species of enteric bacteria, including Escherichia coli. Both probes are able to detect S. hyodysenteriae in colony blot hybridizations, and when applied to fecal specimens, they can detect 10(4) S. hyodysenteriae cells in 0.1 g of seeded fecal matter. Both probes can detect S. hyodysenteriae in fecal specimens from swine with clinical signs of swine dysentery after experimental challenge and from swine from a herd with an acute outbreak of swine dysentery. These probes have application as a diagnostic tool in veterinary microbiology.     

Monoclonal antibodies to a 16-kDa antigen of Serpulina (Treponema) hyodysenteriae.

Monoclonal antibodies (MAbs) were produced to an outer-envelope preparation from Serpulina (Treponema) hyodysenteriae, the aetiological agent of swine dysentery. Three MAbs (isotype IgG1) were obtained. All three recognised a 16-kDa antigen that was common to most strains of S. hyodysenteriae of different serotypes but was absent from nonpathogenic, porcine intestinal spirochaetes. Immunofluorescence and immunogold labelling studies showed that the 16-kDa antigen was exposed on the surface of intact spirochaetes. The MAbs agglutinated freshly grown cultures of spirochaetes and inhibited growth of S. hyodysenteriae strains in vitro.     

Pathogenicity of porcine intestinal spirochetes in gnotobiotic pigs.

Twelve intestinal spirochete strains of porcine origin were characterized on the basis of their phenotypic properties, by multilocus enzyme electrophoresis, and by pathogenicity testing in gnotobiotic pigs. The spirochetes used included two strains of Serpulina hyodysenteriae (B204 and P18A), two strains of Serpulina innocens (B256 and 4/71), one strain from the proposed new genus and species "Anguillina coli" (P43/6/78), and seven non-S. hyodysenteriae strains recently isolated from United Kingdom pig herds with a history of nonspecific diarrhea and typhlocolitis. By multilocus enzyme electrophoresis, five of these were identified as S. innocens, one was identified as an unspecified Serpulina sp., and one was identified as "A. coli." S. hyodysenteriae B204 and P18A, "A. coli" P43/6/78 and 2/7, and three (22/7, P280/1, and 14/5) of the five S. innocens field isolates induced mucoid feces and typhlocolitis in gnotobiotic pigs. None of the other spirochetes produced clinical signs or large intestinal pathology in this model. The "A. coli" strains induced a more watery diarrhea, with lesions present more proximally in the large intestine, than did the other pathogenic spirochetes. S. innocens 22/7 was also tested for pathogenicity in hysterotomy-derived pigs that had previously been artificially colonized with a spirochete-free intestinal flora and shown to be susceptible to swine dysentery. Despite effective colonization, strain 22/7 did not produce any disease, nor was there any exacerbation of large intestinal pathology or clinical signs when pigs with an experimentally induced existing colitis caused by Yersinia pseudotuberculosis were superinfected with strain 22/7. Certain non-S. hyodysenteriae spirochetes are therefore capable of inducing disease in gnotobiotic pigs, but their role as primary or opportunistic pathogens in conventional pigs remains equivocal.     

Analyses of gonococcal lipopolysaccharide in whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: stable association of lipopolysaccharide with the major outer membrane protein (protein I) of Neisseria gonorrhoeae.

The lipopolysaccharide (LPS) of Neisseria gonorrhoeae whole-cell lysates and proteinase K-digested lysates was examined and compared with purified homologous LPS by a method which preferentially stains LPS in polyacrylamide gels. The silver-stained profile of gonococcal LPS in the proteinase K-digested lysate was similar to that of homologous purified LPS; however, the LPS profile in whole-cell lysates was much smaller than that of digested lysates or purified LPS. Conditions of solubilization did not affect these differences. Since it is known that LPS migrates in a unique fashion in second-dimension electrophoresis, the location of LPS in the whole-cell lysates was probed by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a variety of stains and radiolabels. Results from these experiments indicated a stable and reproducible association of LPS with proteins ranging between 23,000 to 36,000 in Mr, in particular major outer membrane protein I. In addition to staining with the silver method, which preferentially stains LPS, the putative LPS was resistant to digestion by proteinase K, did not stain with Coomassie brilliant blue, and was not labeled extrinsically with 125I (Iodogen method) or intrinsically with [35S]methionine. Analysis of two-dimensional gels by immunoblotting with rabbit antisera prepared from protein I bands removed from a polyacrylamide gel revealed the presence of antigens in the same area of the gel (below proteins that were 23,000 to 36,000 in Mr). Antibodies to constituents which migrated below the diagonal were essentially removed by adsorption of antisera with purified LPS, as were antibodies to homologous LPS and LPS in proteinase K-digested whole-cell lysates. Immunoblotting with a monoclonal antibody specific for LPS demonstrated reactivity of the antibody with LPS and with the protein I band. On the basis of these data, we conclude that protein I and perhaps other proteins in the whole-cell lysate are stably associated with LPS; this complex is resistant to dissociation in sodium dodecyl sulfate at high temperature (approximately 100 degrees C) but does, for unknown reasons, dissociate with electrophoresis in the second dimension. The association of LPS with protein antigens in sodium dodecyl sulfate-polyacrylamide gels adds another dimension of complexity to analysis of these antigens by immunoelectroblotting. Furthermore, the tight association of LPS with the major outer membrane protein I may alter the nature of the immune response generated by "purified" protein I vaccine antigens. The possible role of protein-LPS complexes in the pathogenesis of gonorrhea is discussed.     

Lipooligosaccharides from Treponema hyodysenteriae and Treponema innocens.

Lipooligosaccharides from Treponema hyodysenteriae serotypes 1 through 7, attenuated T. hyodysenteriae serotypes 1 and 2, and five strains of T. innocens were extracted with hot phenol water. The extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and analyzed by lipopolysaccharide selective silver staining and Western blot (immunoblot) immunodetection. Silver staining revealed the presence of two bands that ranged between 18,000 and 24,000 daltons and that were serotype specific for T. hyodysenteriae. Attenuation of pathogenic strains resulted in the loss of the higher-molecular-weight band. Four of five T. innocens strains also lacked this particular band. T. innocens 421 had six bands between 17,000 and 26,900 daltons. Western blots with hyperimmune rabbit sera and convalescent-phase swine sera revealed antigenic variation among serotypes of T. hyodysenteriae and attenuated serotypes of T. hyodysenteriae. Convalescent-phase swine sera failed to recognize lipopolysaccharides from T. innocens. Differences in results obtained by lipopolysaccharide selective silver staining versus immunoblotting of the lipopolysaccharide preparations probably indicate that these two methods identify separate characteristics of the same molecule.     

Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization.

Chemotaxis of porcine spirochetes towards a variety of mucins was measured quantitatively by a capillary method. A chemotaxis buffer consisting of 0.01 M potassium phosphate buffer (pH 7.0) and 0.2 mM L-cysteine hydrochloride was necessary for chemotaxis of spirochetes. The optimum incubation time and incubation temperature were 1 h and 40 degrees C, respectively. The mucin concentration also affected the chemotaxis observed, and a concentration of 1% (wt/vol) was near the optimum. Virulent Serpulina hyodysenteriae strains were chemotactic towards 1% (wt/vol) hog gastric mucin and 1% (wt/vol) porcine colonic mucin but not towards 1% (wt/vol) bovine submaxillary mucin. Virulent S. hyodysenteriae strains were significantly more chemotactic than avirulent strains of S. hyodysenteriae (SA3 and VS1), Serpulina intermedius, and Serpulina innocens. Other spirochetes belonging to the proposed group of spirochetes Anguillina coli were also not chemotactic. Pathogenicity of S. hyodysenteriae strains that cause swine dysentery may, in part, be attributed to their attraction to porcine intestinal mucus.     

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.     

A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae.

Serpulina (Treponema) hyodysenteriae P18A and VS1 were extracted by using the detergent Triton X-114 and separated into detergent and aqueous phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis confirmed that a membrane-associated 16-kDa antigen was hydrophobic, since it was found in the detergent phase. A 45-kDa antigen partitioned into the aqueous phase, suggesting that it was hydrophilic and may be of periplasmic origin. When spirochetes were grown in the presence of [3H]palmitic acid, a predominant 16-kDa antigen was labeled; from the results of immunoprecipitation experiments, this antigen appeared to be the same as that recognized by both polyclonal and monoclonal antisera to a previously described 16-kDa antigen. This antigen was proteinase K sensitive and was not a component of the lipopolysaccharide, which, although [3H]palmitate labeled, was resistant to proteinase K digestion. The most probable explanation is that the 16-kDa antigen is a membrane-associated, surface-exposed, immunodominant lipoprotein.     

Characterization of the major outer membrane antigens of Treponema hyodysenteriae.

Outer membrane extracts of Treponema hyodysenteriae were used to evaluate the antibody responses in immunized or convalescent pigs. Western blot (immunoblot) analysis identified antibodies in sera reactive with 14- to 19-kilodalton (kDa) antigens. Reactivity against these antigens could be removed only by absorption of sera with butanol-water-extracted endotoxin from the homologous strain of T. hyodysenteriae. Treatment of the outer membrane extracts with 0.1 M sodium meta-periodate, but not with proteinase K, abolished reactivity with both outer membrane and endotoxin antigens (14 and 19 kDa). These results indicate that swine vaccinated with the outer membrane extract of T. hyodysenteriae develop antibody responses to outer membrane antigens qualitatively similar to those of swine convalescing from active infection, especially antibodies against low-molecular-mass antigens. The nature of the 14- to 19-kDa antigens appears consistent with that of treponemal endotoxin and lipopolysaccharide.     

Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens.

Eleven strains of Treponema hyodysenteriae isolated from pigs with swine dysentery were examined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. T. hyodysenteriae strains formed a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. However, immunoblotting with antiserum from rabbits immunized with T. hyodysenteriae CN8368 revealed heterogeneity among the lipopolysaccharide complexes of different strains. Polypeptides of molecular weights between 30,000 and 36,000 were the predominant T. hyodysenteriae polypeptides detected by porcine immune serum. In contrast, Treponema innocens did not form a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. Adsorption studies and immunoblotting identified polypeptide antigens present on cells of T. hyodysenteriae which were not detected on cells of T. innocens. These unique antigens may play a role in the virulence of T. hyodysenteriae.     

Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the flaB1 gene encoding a 38-kilodalton flagellar protein.

Extracytoplasmic proteins were released from Serpulina (Treponema) hyodysenteriae (strain B204) by treatment of whole cells with a nonionic detergent (Tween 20). Centrifugation of the Tween 20-released proteins at 100,000 x g sedimented 10 major extracytoplasmic proteins with approximate molecular masses of 44, 43.5, 42, 39, 38, 34, 33.5, 33, 31, and 29 kDa. Treatment of the sedimented fraction with 6 M urea solubilized all of the proteins except the 39-kDa protein. Peptide sequences were obtained for the purified 42-, 39-, 38-, 34-, 31-, and 29-kDa proteins. The peptide sequences of the 42-, 38-, and 31-kDa proteins indicate that they likely are components of the periplasmic flagella. The amino-terminal peptide sequence of the 38-kDa protein was used to design an oligonucleotide probe and to clone an S. hyodysenteriae DNA fragment containing the gene encoding this protein. The predicted 290-amino-acid protein sequence derived from the cloned gene was highly homologous to those of several other bacterial flagellar proteins and is preceded by consensus sigma D nucleotide sequences found upstream of other flagellar genes. On the basis of its similarity to the FlaB proteins of other spirochetes, we propose to designate the cloned S. hyodysenteriae gene flaB1 and its encoded protein FlaB1. Vaccination of pigs with FlaB1 or its recombinant counterpart did not protect them from an experimental challenge.     

Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis.

Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species.     

Identification of a new intestinal spirochete with pathogenicity for chickens.

Two intestinal spirochete isolates obtained from chickens with diarrhea were examined by electron microscopy, biochemical tests, rRNA gene restriction pattern analysis, and multilocus enzyme electrophoresis. One isolate (strain 91-1207/C1) was pathogenicity tested in vivo in chickens. The chicken spirochetes were morphologically indistinguishable from Serpulina innocens and Serpulina hyodysenteriae and phenotypically similar to S. innocens. However, the chicken spirochetes could be distinguished from S. innocens, S. hyodysenteriae, and other swine intestinal spirochetes by rRNA gene restriction pattern analysis and multilocus enzyme electrophoresis. In pathogenicity tests in 1-day-old chicks and 14-month-old hens, chicken spirochete 91-1207/C1 produced pale-yellow, watery cecal contents and mild lymphocytic typhlitis. These findings support the conclusion that avian intestinal spirochetes can be pathogenic to commercial poultry and that the microorganisms are different from intestinal spirochetes that infect pigs.     

Monoclonal antibodies against Pseudomonas aeruginosa outer membrane antigens: isolation and characterization.

Hybridomas secreting monoclonal antibodies specific for Pseudomonas aeruginosa outer membrane antigens were isolated. One of the antibodies was highly specific for the O antigen of the lipopolysaccharide of International Antigen Typing Scheme serotype 5 strains, reacting only weakly with a serotype 17 strain and failing to react with the outer membranes of strains representing 15 other serotypes. This monoclonal antibody was able to agglutinate heat-killed bacterial cells as well as lipopolysaccharide-coated sheep erythrocytes. Two other monoclonal antibodies were able to interact with the outer membranes of strains representing all 17 serotypes, although they were unable to agglutinate heat-killed bacterial cells. One of these was shown to be specific for the major outer membrane lipoprotein H2. The antigenic site against which this monoclonal antibody reacted was present in the outer membranes of two Pseudomonas fluorescens strains, two Pseudomonas putida strains, a Pseudomonas anguilliseptica strain, and an Azotobacter vinelandii strain, but not in the outer membranes of five other bacterial species.