Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance.
(9/81)To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml. The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204. Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala. When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer. (+info)
Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9.
(10/81)The study described here was carried out to further characterize reference strains of Serpulina (Treponema) hyodysenteriae representing serotypes 8 and 9. Results obtained from restriction fragment length polymorphism analysis, enteropathogenicity testing, and endotoxin profiles confirmed their identifications. Electron microscopy indicated that both strains were covered with a thin layer of capsule-like material. Immunoblot analysis indicated that an antigen in the 19-kDa region of proteinase K-digested whole cells reacted only with homologous antiserum. The serotype-specific antigens were sensitive to periodate oxidation but resistant to proteinase K digestion and migrated in the same region as purified lipopolysaccharides. Immunoblotting with proteinase K-digested whole cells appeared as useful as immunodiffusion with extracted lipopolysaccharide for the serological classification of S. hyodysenteriae. Immunogold labeling of whole cells and purified periplasmic flagella showed strong cross-reactions between S. hyodysenteriae and Serpulina innocens. Outer membrane preparations of strains representing serotypes 8 and 9 contained four major proteins which reacted with antisera against both species, and one major protein with a molecular mass of 46 kDa which reacted only with antisera against S. hyodysenteriae, irrespective of the serotype. Our findings suggest that periplasmic flagella and some outer membrane proteins are antigens common to both S. hyodysenteriae and S. innocens, whereas a 46-kDa outer membrane protein may be a species-specific antigen of S. hyodysenteriae. Finally, we propose immunoblotting as an alternative method to immunodiffusion for the serotyping of S. hyodysenteriae. (+info)
Characterization of Dutch porcine Serpulina (Treponema) isolates by restriction endonuclease analysis and DNA hybridization.
(11/81)Genomes of 55 Dutch porcine Serpulina (Treponema) hyodysenteriae and non-pathogenic Serpulina isolates were characterized by restriction endonuclease analysis (REA) and DNA hybridization. The Dutch porcine isolates were compared with American Type Culture Collection (ATCC) strains of S. hyodysenteriae and S. innocens and isolates of S. hyodysenteriae with known serotypes (reference strains). REA of the Dutch S. hyodysenteriae isolates resulted in two main patterns, while the non-pathogenic isolates had many distinct REA patterns, all different from the S. hyodysenteriae strains. The S. hyodysenteriae reference strains all had distinct REA patterns, different from the Dutch strains. Upon Southern hybridization with a S. hyodysenteriae DNA fragment encoding a flagellar protein, all S. hyodysenteriae strains could be divided in two groups. The non-pathogenic Serpulina strains had many distinct hybridization patterns and hybridized less intensely. Upon hybridization with a S. hyodysenteriae DNA fragment encoding a haemolysin, DNA of all S. hyodysenteriae strains reacted in the same band. DNA of non-pathogenic Dutch Serpulina strains and S. innocens did not hybridize. It was concluded that there are two main genotypes of S. hyodysenteriae in the Netherlands. This could be of importance for recombinant DNA vaccine development. (+info)
The serological grouping system for Serpulina (Treponema) hyodysenteriae.
(12/81)Lipopolysaccharide from serostrains of Serpulina (Treponema) hyodysenteriae for serogroups A to I was characterized using sodium dodecylsulphate polyacrylamide gel electrophoresis and silver staining. All strains had lipopolysaccharide components ranging from 10 to 16 kDa that represented lipid A-core polysaccharide regions, and short O-antigen side chain were also recognized in certain immunoblots. Serological reactions between lipopolysaccharide and antisera against each of these serostrains were examined by Western immunoblotting. There was relatively little antigenic cross-reactivity between LPS from the nine strains, thus confirming their suitability as serostrains. Using cross-absorbed sera, isolates within serogroups A and E were shown to possess unique epitopes on the core lipopolysaccharide, distinct from serogroup reactivities. These isolates were therefore identified as serovars within the serogroups. This study confirmed the usefulness of the serotyping scheme for S. hyodysenteriae, in which the bacteria can be placed into serogroups using unabsorbed sera, and into serovars within these using cross-absorbed sera. (+info)
The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.
(13/81)The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes. (+info)
Differences in lymphocyte subpopulations and cell counts before and after experimentally induced swine dysentery.
(14/81)The aim of this study was to examine the levels of circulating leukocytes and lymphocyte subpopulations before and immediately after experimentally induced swine dysentery. Twenty-one healthy crossbred pigs (approximately 22 kg) were orally inoculated with Brachyspira hyodysenteriae. Blood was sampled before inoculation and when clinical signs of swine dysentery occurred. Pigs that remained healthy were sampled when killed. Total and differential white blood cell counts were performed, and lymphocyte subpopulations were analysed using flow cytometry. Following a mean incubation period of 13 days, 12 pigs developed swine dysentery, whereas nine remained healthy throughout the study. Before inoculation, pigs that subsequently developed swine dysentery displayed higher levels of circulating gamma delta T cells (mean +/- se; 30.7 +/- 3.5 %) compared with pigs that remained healthy (14.9 +/- 1.4 %). Sick animals also displayed lower levels of CD8 cells (24.6 +/- 1.5 %), cytotoxic/suppressor T cells (10.9 +/- 1.3 %) and CD4 CD8 T cells (8.1 +/- 1.0 %) than the pigs that remained healthy (34.9 +/- 3.1 %; 17.6 +/- 2.0 %; 13.6 +/- 2.3 %). No difference was observed in leukocyte counts before inoculation. At onset of swine dysentery, there was an increase in monocytes (from 1.5 +/- 0.2 x 10 to 3.8 +/- 0.5 x 10 l) and CD4 CD8 T cells (from 5.8 +/- 0.9 to 8.9 +/- 0.7 %). In conclusion, gamma delta T cells and CD8 cells may be associated with susceptibility to experimentally induced swine dysentery, whereas monocytes and CD4 CD8 T cells appear to be the major responding leukocytes during the disease. (+info)
Experimental swine dysentery: comparison between infection models.
(15/81)The aim of the present study was to develop a reproducible porcine infection model with Brachyspira hyodysenteriae. The influence of different factors was evaluated, namely, age, a diet containing large quantities of soybean meal, housing and administration of cortisol or antacids. Furthermore, the synergistic effect of additional bacteria (Escherichia coli O141, Bacteroides vulgatus or a mixture of Bacteroides fragilis, a field isolate of Bacteroides and Fusobacterium necrophorum) was studied. Experimental infection resulted in an increase in the serum concentrations of the acute-phase proteins serum amyloid A and haptoglobin and the percentages of neutrophils and monocytes. These alterations were specifically related to haemorrhagic diarrhoea. Inoculation combined with feeding of large quantities of soybean meal and group-housing induced swine dysentery in all experimental animals. If the pigs were fed soybean meal, kept in single pens and circulated between the pens, five out of nine developed disease. (+info)
Further characterization of porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to tiamulin.
(16/81)Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe diarrhoeal disease in pigs. Few drugs are available to treat the disease, owing to both antimicrobial resistance and withdrawal of drugs authorized for use in pigs. Tiamulin is the drug of choice in many countries, but isolates with decreased susceptibility have recently been reported. The mechanism of tiamulin resistance in B. hyodysenteriae is not known and this facet is essential to understand the dissemination of the trait. To study the resistance epidemiology of B. hyodysenteriae, further characterization of a set of isolates from Germany (n = 16) and the UK (n = 6) with decreased susceptibility to tiamulin was performed. The relatedness between the isolates was studied by comparing PFGE patterns, and the in vitro susceptibility to five other antimicrobials (aivlosin, doxycycline, salinomycin, chloramphenicol and avilamycin) was also determined. For comparison of the antimicrobial-susceptibility pattern, Swedish (n = 20) and British (n = 4) tiamulin-susceptible isolates were tested. The German isolates represented several different PFGE patterns, indicating that tiamulin usage has been sufficient to select clones with decreased tiamulin susceptibility at different farms in Germany. The PFGE pattern for the six British isolates with decreased tiamulin susceptibility was identical to that of the German isolates, and they had a similar antimicrobial-susceptibility pattern, except for resistance to aivlosin, which was only found in a few German isolates. No other co-resistance with tiamulin was found. (+info)