Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.
(49/81)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. (+info)
Production of an inducible sucrase activity by Serpulina hyodysenteriae.
(50/81)Strains of Serpulina hyodysenteriae and Serpulina innocens produced a cell-associated sucrase activity when grown in a medium containing sucrose. S. hyodysenteriae B204 sucrase activity cleaved sucrose and, to a lesser extent, raffinose and had a pH optimum of 5.7 to 6.2. This is the first report of an inducible enzyme produced by either S. hyodysenteriae or S. innocens. (+info)
Evaluation of microagglutination test for differentiation between Serpulina (Treponema) hyodysenteriae and S. innocens and serotyping of S. hyodysenteriae.
(51/81)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. (+info)
Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization.
(52/81)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. (+info)
Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.
(53/81)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. (+info)
Genetic similarity of intestinal spirochetes from humans and various animal species.
(54/81)The chromosomal DNA of spirochetes isolated from human, swine, dog, mouse, rat, and chicken intestine or feces was subjected to restriction enzyme analysis and hybridization with three different DNA probes, derived from a flagellin gene, a hemolysin gene, and the 16S rDNA sequence of the pathogenic swine intestinal spirochete Serpulina hyodysenteriae. This genetic analysis showed that intestinal spirochetes represent a heterogeneous but related population of bacteria. In general, unique genotypes were distinguished among isolates from the same host species; they were not present among isolates from other host species. This suggests the host specificity of some strains. An exception to this are isolates from humans and dogs suffering from gastrointestinal disorders; these isolates showed highly similar or even identical genotypes. None of them resembled any of the genotypes of isolates found in other host species without apparent disease. (+info)
Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.
(55/81)A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes. (+info)
Reduced virulence of Serpulina hyodysenteriae hemolysin-negative mutants in pigs and their potential to protect pigs against challenge with a virulent strain.
(56/81)The role of the Serpulina hyodysenteriae hemolysin encoded by the tlyA gene in the pathogenesis of swine dysentery (SD) was studied. tlyA mutants of two S. hyodysenteriae strains (B204 and C5) were tested for virulence in pigs. None of the animals developed SD. However, after infection with wild-type strain B204 or C5, the incidence of SD was 100 or 60%, respectively. Thus, the tlyA-encoded hemolysin of S. hyodysenteriae is an important virulence factor in SD. The potential of these mutants to protect pigs against challenge with a virulent S. hyodysenteriae strain was also studied. After challenge with wild-type strain B204, 50% of pigs previously inoculated with the B204 tlyA mutant were protected, whereas all control pigs contracted SD. None of the pigs previously inoculated with the C5 tlyA mutant developed SD upon challenge with wild-type strain B204, whereas 40% of the control pigs developed SD in this experiment. Thus, previous colonization with S. hyodysenteriae tlyA mutants in pigs provides partial protection against challenge with a virulent S. hyodysenteriae strain. (+info)