The role of spirochetes in periodontal disease.
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Adult↗
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Major antigens of human oral spirochetes associated with periodontal disease.
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Adolescent↗
Use of commercial enzyme kits and fatty acid production for the identification of Serpulina hyodysenteriae: a potential misdiagnosis.
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.
Animals↗
Laboratory identification and enteropathogenicity testing of Serpulina pilosicoli associated with porcine colonic spirochetosis.
Pathogenic intestinal spirochetes of swine include Serpulina hyodysenteriae, a strongly beta-hemolytic spirochete that causes swine dysentery, and S. pilosicoli, a weakly beta-hemolytic intestinal spirochete (WBHIS) that causes porcine colonic spirochetosis. Because of the existence of nonpathogenic WBHIS in the normal swine colon, it is important to develop laboratory procedures for accurate identification of S. pilosicoli. The purpose of the present study was to assess hippurate hydrolysis and polymerase chain reaction (PCR) amplification of specific 16S ribosomal RNA (rRNA) sequences for identification of porcine S. pilosicoli. Additionally, the enteropathogenicity of 8 field isolates of porcine S. pilosicoli was determined by challenge exposure of 1-day-old chicks and sequential histologic examination of the cecal mucosa. The field isolates of porcine S. pilosicoli hydrolyzed hippurate and yielded S. pilosicoli-specific products by PCR amplification of 16S rRNA sequences. Although all of the field isolates of porcine S. pilosicoli attached to the cecal epithelium of challenge-exposed chicks by day 21 postinoculation, some isolates had locally invasive phenotypes. We concluded that identification of porcine S. pilosicoli could be made on the basis of results of hippurate hydrolysis and 16S rRNA PCR amplification. Challenge inoculation of 1-day-old chicks followed by histologic examination of the cecal mucosa demonstrated the enteropathogenicity of porcine S. pilosicoli.
Animals↗
Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens.
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.
Animals↗
In vitro activity of four antimicrobial agents against North American isolates of porcine Serpulina pilosicoli.
Porcine colonic spirochetosis is a nonfatal diarrheal disease that affects pigs during the growing and finishing stages of production. The disease is caused by Serpulina pilosicoli, a newly recognized species of pathogenic intestinal spirochete. Antimicrobial therapy aimed at reducing the infection may be helpful in controlling spirochetal diarrhea. In this study, the in vitro antimicrobial susceptibilities of the reference isolate S. pilosicoli P43/6/78 from the United Kingdom and 19 field isolates obtained from pigs in Canada (n = 5) and the United States (n = 14) were determined against the antimicrobial agents carbadox, gentamicin, lincomycin, and tiamulin, all of which are commonly used for control of the related pathogenic intestinal spirochete S. hyodysenteriae. Additionally, the susceptibility or resistance of each isolate against each antimicrobial agent was estimated on the basis of available data on the in vitro antimicrobial susceptibility breakpoints of S. hyodysenteriae. Each isolate was identified on the basis of phenotypic and genotypic markers, and the minimum inhibitory concentration of each antimicrobial agent was determined by the agar-dilution method. All the isolates were susceptible to carbadox and tiamulin. The percentages of isolates susceptible, intermediate, and resistant to lincomycin were 42.1%, 42.1%, and 15.8%, respectively. Slightly less than half of the isolates (47.4%) were susceptible to gentamicin, and the remainder (52.6%) were resistant. Implementation of rational control measures to reduce infection by S. pilosicoli should improve overall health and productivity in swine herds.
Animal Husbandry↗
Minimum inhibitory concentration of selected antimicrobial agents for Serpulina isolated from chickens and rheas.
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Animals↗
Periodontitis vs. peri-implantitis: the same disease? The same treatment?
The microbial flora in the natural dentition sulcus/pocket and the implant crevice/pocket is very similar in both health and disease. In health, coccal forms predominate, and in disease, large numbers of Gram-negative pathogens are associated with both tooth and implant. It has also been demonstrated that the bacteria in the partially edentulous implant case may be more pathogenic (especially Gram-negative rods and spirochetes) than in the fully edentulous case, indicating a possible seeding mechanism from tooth pocket to implant crevice. Detoxification procedures involving the use of tetracycline and citric acid prior to regenerative procedures with the use of barrier membranes and grafting materials are necessary, and the same problems attendant to premature exposure of the barrier membrane(s) in the natural dentition situation apply to the implant case. It is apparent that periodontitis = peri-implantitis in etiology and therapy.
Anti-Bacterial Agents↗
Spirochete-platelet attachment and thrombocytopenia in murine relapsing fever borreliosis.
Thrombocytopenia is common in persons infected with relapsing fever Borreliae. We previously showed that the relapsing fever spirochete Borrelia hermsii binds to and activates human platelets in vitro and that, after platelet activation, high-level spirochete-platelet attachment is mediated by integrin alpha IIb beta 3, a receptor that requires platelet activation for full function. Here we established that B hermsii infection of the mouse results in severe thrombocytopenia and a functional defect in hemostasis caused by accelerated platelet loss. Disseminated intravascular coagulation, immune thrombocytopenic purpura, or splenic sequestration did not play a discernible role in this model. Instead, spirochete-platelet complexes were detected in the blood of infected mice, suggesting that platelet attachment by bacteria might result in platelet clearance. Consistent with this, splenomegaly and thrombocytopenia temporally correlated with spirochetemia, and the severity of thrombocytopenia directly correlated with the degree of spirochetemia. Activation of platelets and integrin alpha IIb beta 3 were apparently not required for bacterium-platelet binding or platelet clearance because the bacterium-bound platelets in the circulation were not activated, and platelet binding and thrombocytopenia during infection of beta 3-deficient and wild-type mice were indistinguishable. These findings suggest that thrombocytopenia of relapsing fever is the result of platelet clearance after beta 3-independent bacterial attachment to circulating platelets.
Animals↗
Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection.
A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.
Animals↗
Bacterial alpha2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?
BACKGROUND: Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor alpha2-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion. RESULTS: Database searches with metazoan alpha2-macroglobulin sequences revealed homologous sequences in bacterial proteomes. The bacterial alpha2-macroglobulin phylogenetic distribution is patchy and violates the vertical descent model. Bacterial alpha2-macroglobulin genes are found in diverse clades, including purple bacteria (proteobacteria), fusobacteria, spirochetes, bacteroidetes, deinococcids, cyanobacteria, planctomycetes and thermotogae. Most bacterial species with bacterial alpha2-macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts. Both pathogenically invasive and saprophytically colonizing species possess bacterial alpha2-macroglobulins, indicating that bacterial alpha2-macroglobulin is a colonization rather than a virulence factor. CONCLUSIONS: Metazoan alpha2-macroglobulins inhibit proteases of pathogens. The bacterial homologs may function in reverse to block host antimicrobial defenses. Alpha2-macroglobulin was probably acquired one or more times from metazoan hosts and has then spread widely through other colonizing bacterial species by more than 10 independent horizontal gene transfers. yfhM-like bacterial alpha2-macroglobulin genes are often found tightly linked with pbpC, encoding an atypical peptidoglycan transglycosylase, PBP1C, that does not function in vegetative peptidoglycan synthesis. We suggest that YfhM and PBP1C are coupled together as a periplasmic defense and repair system. Bacterial alpha2-macroglobulins might provide useful targets for enhancing vaccine efficacy in combating infections.
Alphaproteobacteria↗
Effect of terdecamycin on experimentally induced swine dysentery in pigs.
The authors evaluated the effect of terdecamycin, a novel antibiotic, on experimentally induced Serpulina (S.) hyodysenteriae infection in pigs. In a prophylactic test, feed containing terdecamycin was fed to pigs for 7 days before inoculation and 21 days after inoculation. Dysenteric diarrhea, development of lesions in the large intestinal mucosa and colonization of S. hyodysenteriae in colonic mucosa were completely inhibited by treatment with 5 ppm or 10 ppm terdecamycin. In the therapeutic test, an unmedicated feed was fed to pigs inoculated with S. hyodysenteriae, until the typical mucohemorrhagic diarrhea appeared after inoculation, and was then changed to the medicated feed for 10 days. By treating with 20 ppm terdecamycin, the clinical signs in the pigs were improved and S. hyodysenteriae was completely eradicated.
Animals↗
Presence of 22- and 17-kDa proteins reacting with sera in mice experimentally infected with Brachyspira (Serpulina) hyodysenteriae.
The antibodies to B. (S.)hyodysenteriae in experimentally infected mice were detected by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). The reactions in MAT were serotype specific while those in ELISA were common to both strains. A further investigation with immunoblotting technique demonstrated that 22- and 17-kDa proteins reacted strongly with the sera. The proteins in ATCC 27164 strain strongly reacted with the serum from ATCC 31212 strain-infected mouse and vice versa. These proteins were sensitive to proteinase K.
Animals↗
Intestinal spirochetosis in wild sika deer (Cervus nippon yesoensis) infected with Brachyspira species.
Seven adult free-ranging sika deer (Cervus nippon yesoensis) were examined by histology, immunohistochemistry and electron microscopy for intestinal spirochetal infection. Histologically epithelial and goblet cell hyperplasia and edema of the lamina propria mucosa with macrophage and lymphocyte infiltration were observed in the cecum and colon in 6 of the 7 deer. Numerous argyrophilic spirochetes were present in the crypts and some had invaded epithelial and goblet cells and caused degeneration. Immunohistochemically the organisms stained positively with polyclonal antisera against Brachyspira (Serpulina) hyodysenteriae and B. pilosicoli. Ultrastructurally they were 6-14 microm long, 0.2-0.3 microm wide and had 4-6 coils and 13 axial filaments per cell; such features were closely similar to those in the Brachyspira species. These results showed that the spirochetes were capable of inducing enteritis in deer and this intestinal spirochete infection might already be prevalent among wild sika deer in Japan. There is a possibility that this spirochetal colitis is a new syndrome in sika deer and that the same and/or similar spirochetes have infected ruminants, including sika deer and cattle.
Animals↗
In vitro susceptibility and a new point mutation associated with tylosin-resistance in Japanese canine intestinal spirochetes.
The in vitro susceptibilities of six commonly used antimicrobial agents against 29 isolates of intestinal spirochetes isolated from dogs in Japan were examined by the agar dilution technique. In addition, the genetic basis of tylosin resistance in in vitro selected resistant mutants of two reference strains and three tylosin-susceptible field isolates obtained by three successive subcultures on blood agar containing 1 microg/ml of tylosin was investigated. Carbadox was the most active (MIC: < 0.00625) of all the antimicrobial agents. Although all the isolates were susceptible to tylosin, some were resistant to erythromycin. Tiamulin, lincomycin and dimetridazole were also very active against the isolates. All the resistant isolates did not harbor any plasmids. In vitro selected tylosin-resistant mutants of previously tylosin-susceptible isolates showed a new mutation in which their adenine at the base position equivalent to 2062 of 23S rDNA of Escherichia coli has been replaced by cytosine. These findings may both provide guidance towards the proper choice of antimicrobial agents for the treatment of canine intestinal spirochetosis, and add to the understanding of the genetic basis of tylosin resistance.
Animals↗
A possibility of application of the 105-kilodaltons protein of Brachyspira alvinipulli cross-reacting with antisera to five species in the genus Brachyspira to diagnosis.
The antigenic properties of Brachyspira (B.) alvinipulli ATCC 51933 and strain C2 were analyzed and compared with those of B. hyodysenteriae ATCC 27164 and ATCC 31212, B. pilosicoli ATCC 51139, B. innocens ATCC 29796 and B. aalborgi NCTC 11492. In gel immunodiffusion tests, a protein in B. alvinipulli ATCC 51933 reacted strongly with anti-B. alvinipulli ATCC 51933-serum and formed two precipitin lines. Furthermore, by an immunoblotting technique, the 105-kilodaltons (kDa) protein in B. alvinipulli ATCC 51933 reacted strongly with each of the antisera to B. hyodysenteriae, B. pilosicoli, B. innocens and B. aalborgi. Therefore, the 105-kDa protein could be applied to diagnosis of chicken infection by B. alvinipulli and B. pilosicoli. But the 105-kDa protein reacting with the anti-B. alvinipulli ATCC 51933-serum was not confirmed in B. hyodysenteriae, B. pilosicoli, B. innocens and B. aalborgi. The N-terminal amino acid sequence of the 105-kDa protein isolated from B. alvinipulli ATCC 51933 was Met-Lys-Lys-Met-Val-Tyr-Phe-Phe-Gly-Asn. The amino acid alignment of this protein possessed 50% homology with the periplasmic-iron-binding protein BitC in B. hyodysenteriae.
Animals↗
The specific antibody to Brachyspira aalborgi in serum obtained from a patient with intestinal spirochetosis.
Serum obtained from a patient histopathologically diagnosed as intestinal spirochetosis was investigated serodiagnostically by agglutination test. B. aalborgi which is a human intestinal spirochete reacted strongly with the human serum, while B. pilosicoli which has potential pathogenicity to humans reacted with the serum, but as strongly and its titer was different than the other three species. On the other hand, intestinal spirochetes (Matsumoto isolates) were isolated from the biopsy samples of the patient. The morphological, biochemical, and genetic characteristics of the isolates were very similar to those of B. aalborgi. Furthermore, the protein profiles of the Matsumoto isolates were also similar to those of B. aalborgi but were different than those of B. pilosicoli and B. hyodysenteriae. The reaction profiles of the Matsumoto isolates in immunoblotting were relatively similar to those of B. aalborgi except for a 74 kDa band but were different from those of B. pilosicoli and B. hyodysenteriae. Therefore, we identified the Matsumoto isolates as B. aalborgi and diagnosed the patient with a B. aalborgi infection.
Agglutination Tests↗
A comparison of the morphologic effects of Serpulina hyodysenteriae or its beta-hemolysin on the murine cecal mucosa.
Studies were carried out to compare the early morphologic changes in the cecal mucosa of mice either infected with Serpulina hyodysenteriae or exposed to the beta-hemolysin of S. hyodysenteriae. Sixty-five 12-24-week-old C3H/HeOuJ mice were infected with S. hyodysenteriae by gastric intubation. Two mice were necropsied every hour for 30 hours following infection. S. hyodysenteriae was isolated from the cecal contents of each mouse at all time points. Macroscopic lesions were first apparent at 14 hours postinfection (PI), and light microscopic lesions were first apparent at 10 hours PI, earlier than has been previously reported. Ultrastructural changes, first evident at 6 hours PI, included disarray and loss of microvilli and terminal web, with dilatation of intercellular spaces. Luminal bacteria were translocated through epithelial cells to the lamina propria, where capillaries exhibited changes indicative of increased permeability. In another experiment, solutions containing between 2,500 and 25,000 hemolytic units of purified S. hyodysenteriae hemolysin were placed within the lumen of surgically closed murine ceca (n = 10); ceca were collected for examination 3 hours following treatment. Ultrastructural changes consisted of loss of microvilli and terminal web and marked vacuolation and exfoliation of epithelial cells. Significant numbers of necrotic and apoptotic epithelial cells were present, and epithelial cells internalized moderate numbers of bacteria. The hemolysin of S. hyodysenteriae induces some of the same early ultrastructural changes in the cecal epithelium of mice as occur following infection with S. hyodysenteriae. Based on the observed bacterial translocation, luminal bacteria also appear to play a unique role in lesion development in this model.
Animals↗