Production and characterization of monoclonal antibodies specific for lipooligosaccharide of Serpulina hyodysenteriae.
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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. (+info)
Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange.
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Serpulina hyodysenteriae, the etiologic agent of swine dysentery, contains complex periplasmic flagella which are composed of multiple class A and class B polypeptides. To examine the role these proteins play in flagellar synthesis, structure, and function and to develop strains which may provide insight into the importance of motility in the etiology of this pathogen, we constructed specific periplasmic flagellar mutations in S. hyodysenteriae B204. The cloned flaA1 and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and/or kanamycin gene cassettes. Following delivery of these suicide plasmids into S. hyodysenteriae, homologous recombination and allelic exchange at the targeted chromosomal flaA1 and flaB1 genes was verified by PCR, sequence, and Southern analysis. The utility of a chloramphenicol resistance gene cassette for targeted gene disruption was demonstrated and found more amenable than kanamycin as a selective marker in S. hyodysenteriae. Immunoblots of cell lysates of the flagellar mutants with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath or core protein. Both mutations selectively abolished expression of the targeted gene without affecting synthesis of the other flagellar polypeptide. flaA1 and flaB1 mutant strains exhibited altered motility in vitro and were less efficient in movement through a liquid medium. Paradoxically, isogenic strains containing specifically disrupted flaA1 or flaB1 alleles were capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. This is the first report of specific inactivation of a motility-associated gene in spirochetes. (+info)
Pathogenicity of human and porcine intestinal spirochetes in one-day-old specific-pathogen-free chicks: an animal model of intestinal spirochetosis.
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One-day-old chicks were infected orally with two strains of weakly hemolytic spirochetes isolated from a human and a pig with intestinal spirochetosis. These spirochetosis both colonized birds, attached end-on to their cecal enterocytes, induced watery diarrhea, and significantly depressed growth rates. Cultures of Serpulina innocens failed to colonize the chicks. (+info)
Growth of Serpulina (Treponema) hyodysenteriae under iron-restricted conditions.
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Reference strains of Serpulina hyodysenteriae expressed at least three iron-regulated proteins with apparent molecular masses of > 200, 134, and 109 kDa when grown under iron-restricted conditions. Cells of S. hyodysenteriae grown under these conditions also showed increased outer membrane bleb formation when examined by electron microscopy after negative staining. S. hyodysenteriae did not use the 2 most common types of siderophore, namely catechol and hydroxamate. Western blotting with serum from a pig experimentally infected with S. hyodysenteriae B204 indicated that the 109-kDa major iron-regulated protein was expressed in vivo and was conserved among all strains tested. (+info)
Susceptibility of porcine ileal enterocytes to the cytotoxin of Serpulina hyodysenteriae and the resolution of the epithelial lesions: an electron microscopic study.
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The cytotoxin from Serpulina hyodysenteriae was injected into ileal loops of eight germ-free pigs, and the effects on the villi were observed after 1, 3, and 18 hours of exposure. The mature vacuolated villus enterocytes of the proximal part of the absorptive villi were most susceptible to the lethal effects of the cytotoxin and were extensively exfoliated. The enterocytes at the base of the villi, the goblet cells, and the follicle-associated epithelium of the dome villi, particularly the M cells, were less affected. As the enterocytes were shed, the villi progressively shortened and the basement membrane became extensively folded. The absorptive villi were markedly stunted at 3 hours, and flattened globlet cells predominated at the site of restitution of the lesion. The myofibroblasts were also damaged, apparently subsequent to the exfoliation of the enterocytes. There was no further damage at 18 hours. The absorptive villi were stunted and were devoid of the large interstitial spaces of the normal lamina propria; the enterocytes were generally columnar, and at the apex of each villus there was an accumulation of goblet cells. There was a preponderance of M cells at the apices of the dome villi. Restitution of the lesions was not as rapid as observed in in vitro systems. The changes observed indicated that as the proximal enterocytes of the absorptive villi were shed, the loss of hydrostatic forces in the lamina propria allowed the myofibroblasts to collapse the villi by progressively retracting the basement membrane. This reduced the surface area to be covered during restitution. Resolution of the lesions was still incomplete after 18 hours. (+info)
Use of commercial enzyme kits and fatty acid production for the identification of Serpulina hyodysenteriae: a potential misdiagnosis.
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The accuracy of identification of Serpulina hyodysenteriae strains grown in a complex medium was 90% when 2 commercial test kits were used. Unlike the other S. hyodysenteriae strains, S. hyodysenteriae strain P35/2 was unusual in being indole negative. The nonpathogenic intestinal spirochete PWS/A, which is from a different species, was indole positive and alpha-galactosidase negative. Identification of these spirochetes on the basis of these kits alone would have been incorrect. The analysis of volatile fatty acids by gas chromatography showed that the ratio of acetic to butyric acid was from 11:1 to 44:1 for S. hyodysenteriae strains, which distinguished them from the other spirochetes. The exception was PWS/A (acetic: butyric of 32:1), but this spirochete, unlike the S. hyodysenteriae spirochetes, also produced isobutyric acid. Short chain fatty acid (SCFA) analysis by high-performance liquid chromatography detected different SCFAs in addition to acetic and butyric acids. These additional SCFAs did not contribute to further differentiation of the porcine spirochetes. (+info)
Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the flaB1 gene encoding a 38-kilodalton flagellar protein.
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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. (+info)
Morphometric analysis of enteric lesions in C3H/HeN mice inoculated with Serpulina hyodysenteriae serotypes 2 and 4 with or without oral streptomycin pretreatment.
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The segmental distribution and sequential progression and the role of the indigenous bacterial flora in the development of enteric lesions associated with Serpulina hyodysenteriae infection in laboratory mice have not been defined. We examined the distribution and sequential morphometric changes in the large intestine of mice orally inoculated with S. hyodysenteriae serotypes 2 and 4. To determine the role of colonization resistance conferred by the indigenous bacterial flora, 40 female C3H/HeN mice were administered water alone or water containing 5 mg/mL streptomycin sulfate ad libitum for seven days prior to orogastric inoculation either with S. hyodysenteriae or sterile trypticase soy broth (TSB). Clinical signs were monitored daily and three mice per group were necropsied on postinoculation days (PID) 7 and 14 for pathological assessment of the cecum, proximal colon, transverse colon, and descending colon, and bacteriological culture of the cecum for S. hyodysenteriae. Weekly pooled fecal samples were collected from each group for determination of total numbers of anaerobe bacteria. Gross examination revealed soft fecal pellets on PID 7 and 14 and catarrhal typhlitis on PID 14, irrespective of streptomycin pretreatment. The recovery rates of S. hyodysenteriae from the ceca of serotype 2- and serotype 4-inoculated mice was 100 and 91.7%, respectively. Statistically significant differences in morphometric changes between TSB- and S. hyodysenteriae-inoculated mice were present on PID 7 and 14 and were restricted to the cecum. Although oral administration of streptomycin for seven days prior to S. hyodysenteriae inoculation resulted in a significant reduction in the numbers of fecal anaerobes, it did not affect the colonization, distribution, severity, or progression of cecal lesions. (+info)