Development of an experimental model allowing discrimination between virulent and avirulent isolates of Serpulina (Treponema) hyodysenteriae.
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Variation in virulence among different strains of Serpulina hyodysenteriae was studied by oral inoculation of specific pathogen free piglets and CD-1 mice. Piglets infected with serotype 2 reference strain B204 and an untypable field strain LHV-90-9-I had severe diarrhea tainted intermittently with mucus and fresh blood. The piglets inoculated with B169, B8044, B6933, and ACK300-8 reference strains representing serotypes 3, 5, 6, and 7 respectively developed moderate diarrhea. However, reference strains B234 and A-1 of serotypes 1 and 4, respectively, failed to cause any diarrhea. None of the S. hyodysenteriae strains caused diarrhea in mice. The results indicate a great variation in virulence among strains of different serotypes of S. hyodysenteriae. Mice were less susceptible to infection with S. hyodysenteriae. (+info)
The incidence of swine dysentery in pigs can be reduced by feeding diets that limit the amount of fermentable substrate entering the large intestine.
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Two experiments were conducted to test the hypothesis that feeding diets which limit the amount of fermentable substrate entering the large intestine would protect pigs against experimental infection with Serpulina hyodysenteriae, the causative agent of swine dysentery. Experiment 1 examined the effect of grain processing (hammer milling vs. steam flaking) and grain type (barley, groats, corn, sorghum and wheat) on indices of fermentation in the large intestine and the incidence of swine dysentery. Experiment 2 examined the role of five diets, steam-flaked corn, steam-flaked sorghum, hammer-milled wheat, extruded wheat and cooked white rice, on these same measures. All diets contained an animal protein supplement and no antibiotics. Pigs fed diets based on steam-flaked corn and steam-flaked sorghum had a lower incidence of disease (11-33%) than pigs fed diets based on other grains (75-100%). Pigs fed the diet based on cooked white rice were fully protected against swine dysentery. Both the soluble non-starch polysaccharide (NSP) concentration and the total NSP concentration of the diets explained a significant proportion of the variation in swine dysentery (R2 = 0.56, P = 0.016, and R2 = 0.71, P = 0.002, respectively), such that pigs eating diets containing <1.0 g/100 g soluble NSP showed reduced disease. However, pigs fed corn, sorghum and steam-flaked sorghum (Experiment 2), which contained only 0.4-0.5 g/100 g soluble NSP, still had a high incidence of disease (>50%). This was attributable to a higher level of resistant starch present in these grains. These data provide evidence that the expression of swine dysentery is associated with an increased concentration of fermentable substrate entering the large intestine. (+info)
Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery.
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The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus. (+info)
Induction of interleukin (IL)-1beta and IL-8 mRNA expression in porcine macrophages by lipopolysaccharide from Serpulina hyodysenteriae.
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Lipopolysaccharide (LPS) is a classic inducer of inflammatory cytokines and is a key virulence factor for most gram-negative pathogens. The effect of phenol-water (LPS) and butanol-water (endotoxin) extracts from Serpulina hyodysenteriae on inflammatory cytokine mRNA expression from porcine alveolar macrophages was investigated. The LPS and endotoxin extracts from S. hyodysenteriae induced a dose-dependent expression of interleukin 1beta (IL-1beta) and IL-8 which was weak compared with the responses induced by Escherichia coli LPS. In addition, the spirochetal extracts induced no detectable upregulation of mRNA expression for either IL-6 or tumor necrosis factor alpha. (+info)
Enhanced isolation of Serpulina hyodysenteriae by using sliced agar media.
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A method has been developed for separating Serpulina hyodysenteriae, a large spirochete and the causative agent of swine dysentery (SD), from other fecal anaerobic bacteria in rectal and colonic swabs. This was done by cutting the blood agar in parallel cuts and streaking perpendicular to the cuts in the center of the petri dish. Migration of S. hyodysenteriae from the central streak was apparent by the presence of strong beta-hemolysis along the edges of the cuts. If only S. hyodysenteriae migrated in the cut, they migrated to the end of the cut. However, if both motile bacteria and S. hyodysenteriae migrated in the cut, the motile bacteria migrated to the end of the cut where they formed colonies and the S. hyodysenteriae located along the edges of the cut between the colonies of motile bacteria and the central streak. Although motile bacteria were present where S. hyodysenteriae located, the growth of the motile bacteria was partially inhibited since they rarely formed visible colonies and were low in number. The cut in the agar was thought to improve traction for the serpentine movement of the S. hyodysenteriae and for the flagellar movement of the motile bacteria. Use of sliced blood agar was superior to conventionally streaked blood agar in that (i) it was easier to see strong beta-hemolysis on sliced agar; (ii) frequently, a confirmatory diagnosis could be made using only one petri dish with sliced agar, thereby saving time and media; (iii) S. hyodysenteriae could sometimes be isolated free of other bacteria; and (iv) sliced agar was more effective in isolating S. hyodysenteriae from swine with chronic diarrhea and nondiarrhetic carriers of SD in which the shedding of S. hyodysenteriae was low. (+info)
Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae.
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Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete. (+info)
Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.
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Proliferative enteritis, swine dysentery, and porcine salmonellosis are the most common enteric bacterial diseases affecting pigs in the growing and finishing stages of production. Currently, diagnoses of these diseases by standard cultural techniques of intestinal specimens can be laborious, time consuming, and expensive (swine dysentery, porcine salmonellosis) or impossible (proliferative enteritis). Amplification by polymerase chain reaction (PCR) of DNA sequences specific for each bacterial agent is a highly sensitive and specific method that overcomes the limitations associated with standard detection methods. A multiplex PCR (M-PCR) assay was developed for simultaneous detection and identification of the etiologic agents associated with proliferative enteritis, swine dysentery, and porcine salmonellosis in a single reaction using total DNA obtained directly from intestinal specimens. Purified DNA obtained from pure cultures of each bacterial agent alone or mixed in different combinations and concentrations and total DNA from intestinal specimens were amplified using the Lawsonia intracellularis-, Serpulina hyodysenteriae-, and salmonellae-specific M-PCR assay. Intestinal specimens consisted of feces and mucosal scrapings obtained from field cases of each disease alone or in combinations and feces obtained from pigs challenged with S. hyodysenteriae. The banding pattern of the amplified PCR products, after agarose gel electrophoresis and staining, indicated the presence of individual or combinations of etiologic agents in each specimen. Results from this study indicated that simultaneous amplification of L. intracellularis-, S. hyodysenteriae-, and salmonellae-specific DNA sequences by M-PCR can be used for specific detection and identification of three major enteric bacterial pathogens associated with proliferative enteritis, swine dysentery, and porcine salmonellosis occurring alone or in combinations. Also, the M-PCR assay can be done using DNA obtained directly from intestinal specimens submitted for diagnostic investigation. (+info)
Structure-activity relationships of hygromycin A and its analogs: protein synthesis inhibition activity in a cell free system.
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Several analogs of hygromycin A were tested in an Escherichia coli cell free protein synthesis inhibition assay and in a Serpulina hyodysenteriae whole cell assay. The aminocyclitol moiety is essential for antibacterial activity in both cell free and whole cell assays. However a 4'-O-allyl ether of hygromycin A aglycone showed an equivalent MIC to hygromycin A, while having a less potent IC50 in the cell free assay. Hence 6-deoxy-5-keto-D-arabino-hexofuranose can be replaced by a hydrophobic allyl group and still retain antibacterial activity. However, this replacement reduces the intrinsic protein synthesis inhibition activity. The loss of intrinsic activity with replacement by the allyl group may be compensated for by better transport into the bacterial cell. In addition to the SAR analysis, we demonstrated that the ineffectiveness of hygromycin A against Gram-negative enteric bacteria such as Escherichia coli is mainly due to the efflux mechanism (Acr A/B pump) existing widely among the enteric bacteria rather than the impermeable barrier of the outer membrane. (+info)