A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation.

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.

Spirochaeta aurantia has diacetyl chloramphenicol esterase activity.

The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, alpha-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.

Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis.

A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modified transiently--smooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor operation and a motor switch synchronizing device.

Susceptibility of small-sized oral spirochetes to eight antibiotics and chlorhexidine.

The purpose of the study was to examine the susceptibility of two different small-sized spirochete morphotypes from subgingival plaque to eight antibiotics and chlorhexidine. MIC-values were determined by a broth dilution method and related to achievable antibiotic tissue- and blood concentrations. The spirochetes were characterized as susceptible, moderate susceptible, and resistant to the different antibiotics. The MICs of tetracycline hydrochloride, doxycycline, penicillin G, and chlorhexidine were all considerably lower for spirochete strains with a 2:4:2 endoflagella system (two endoflagella from each cell-end) than for strains with a 1:2:1 endoflagella system (one endoflagellum from each cell-end). No differences were observed for the remaining antibiotics. Spirochetes containing one endoflagellum from each cell-end were found to be susceptible to metronidazole and doxycycline, susceptible to moderate susceptible to tetracycline hydrochloride, moderate susceptible to penicillin G, and resistant to the remaining antibiotics. Spirochetes containing two endoflagella from each cell-end were susceptible to doxycycline, tetracycline hydrochloride, and metronidazole. Susceptible to moderate susceptible to penicillin G. These spirochetes were resistant to the remaining agents. The significance of these observations is discussed in relation to obtainable gingival crevicular fluid concentrations and treatment of marginal periodontitis.

Uptake of D-xylose and D-glucose by Spirochaeta aurantia.

Uptake of D-[14C]glucose and D-[14C]xylose by Spirochaeta aurantia was demonstrated to be osmotic shock sensitive and to require a high-energy phosphorylated compound rather than a proton motive force. These features are similar to those of binding protein-mediated transport systems in other gram-negative bacteria.

Selection of ion channel elements in the serine and aspartate methyl-accepting chemotaxis proteins of bacteria.

Two plausible, transmembrane ion channel elements (These 'elements' are alpha-helical sequences of 24 amino acids in which polar, hydrophilic side chains occupy one side and hydrophobic side chains the other) have been identified in the serine chemoreceptor-methyl-accepting chemotaxis protein (MCP) (SerR) of E. coli and the aspartate chemoreceptor-MCP (AspR) of S. typhimurium. That the chemoreceptor might serve as, or activate, an ion channel is supported strongly by the occurrence of membrane depolarization, specific peptide methylation and neurotoxin inhibition of response in the chemotaxis of S. aurantia (E.P. Greenberg, refs. 13-18).

Inhibition of Spirochaeta aurantia chemotaxis by neurotoxins.

The effects of neurotoxic compounds on the chemotactic response of Spirochaeta aurantia were investigated. In the presence of neurotoxins that affect action potential generation and transmission in excitable eucaryotic cells, D-xylose taxis was inhibited by 69 to 93%. Inhibition of chemotaxis was not due to decreased viability or motility. This study supports the hypothesis that the molecular basis for sensory signal transduction in S. aurantia involves ion fluxes across the cytoplasmic membrane.

Meptazinol diminishes the Jarisch-Herxheimer reaction of relapsing fever.

Naloxone, an opioid antagonist, and meptazinol, an opioid antagonist with agonist properties, were tested in a double-blind placebo-controlled trial in 24 Ethiopian patients with louse-borne relapsing fever. The potentially fatal Jarisch-Herxheimer reaction (J-HR), which invariably follows tetracycline treatment of the disease, was unaffected by naloxone, 30-40 mg intravenously, but was diminished by meptazinol, 300-500 mg intravenously. Compared with naloxone and placebo, meptazinol reduced the clinical severity of the reaction, significantly delayed and shortened its chill phase, delayed the rise in temperature, and reduced peak temperature and changes in pulse and respiratory rates and leucocyte count. High-dose corticosteroids given before or at the time of tetracycline treatment failed to alter the reaction, which is thought to result from release of endotoxin-like substances. Meptazinol is the first effective treatment for the J-HR of louse-borne relapsing fever. This finding suggests a role for endogenous opioids in the pathogenesis of the J-HR.

Chemotaxis in Spirochaeta aurantia.

Cell of Spirochaeta aurantia M1 suspended in isotropic buffer solution swam in nearly straight lines and appeared to spin around their longitudinal axis. Occasionally, cells stopped and flexed, and then resumed translational motility, usually in a different direction. The average cell velocity was 26 micron/s. A quantitative assay for chemotaxis was used to test various chemicals for their ability to attract S. aurantia M1. The cells exhibited a tactic response toward 5 X 10(-2) M D-glucose between 10 and 35degree C; the optimum response was at 25degree C. At 5 degree C motility was not impaired, but D-glucose taxis was abolished. Chemotaxis toward D-glucose was stimulated by L-cysteine (2 X 10(-4) M). D-Glucose, 2-deoxy-D-glucose, alpha-methyl-D-glucoside, D-galactose, D-fucose, D-mannose, D-fructose, D-xylose, maltose, cellobiose, and D-glucosamine were effectve attractants for S. aurantia M1. D-Galactose taxis and D-fucose taxis were induced by the presence of D-galactose in the growth medium. The amino acids tested did not serve as attractants, tgrowing cells of S. aurantia M1 exhibited an aerotactic response.

Glucose and pyruvate metabolism of Spirochaeta litoralis, an anaerobic marine spirochete.

The pathways of glucose and pyruvate metabolism in Spirochaeta litoralis, a free-living, strictly anaerobic marine spirochete, were studied. Addition of 0.2 to 0.4 M NaCl (final concentration) to suspending buffers prevented cell lysis and was necessary for gas evolution from various substrates by cell suspensions. The organism fermented glucose mainly to ethanol, acetate, CO(2), and H(2). Determination of radioactivity in products formed from (14)C-labeled glucose and assays of enzymatic activities in cell extracts indicated that S. litoralis catabolized glucose via the Embden-Meyerhof pathway. A clostridial-type clastic reaction was utilized by the spirochete to degrade pyruvate to acetyl-coenzyme A, CO(2), and H(2). Formation of acetate from acetyl-coenzyme A was catalyzed by phosphotransacetylase and acetate kinase. Nicotinamide adenine dinucleotide-dependent acetaldehyde and alcohol dehydrogenases converted acetyl-coenzyme A to ethanol. A reversible hydrogenase activity was detected in cell extracts. S. litoralis cell extracts contained a rubredoxin similar in spectral properties to other bacterial rubredoxins.

Spirochete from the rabbit tick, Haemaphysalis leporispalustris (Packard). I. Isolation and preliminary characterization.

An unidentified spirochete, referred to as the 277F agent, was isolated from Haemaphysalis leporispalustris ticks from two cottontail rabbits by inoculation of the tick suspension into embryonated chicken eggs. Because of its minute width (0.1 mu), the organism was difficult to see when stained by the Giemsa method, but was readily demonstrated by silver impregnation or fluorescent-antibody procedures. In dark-field microscopy, the spirochetes appeared uniformly and rather tightly coiled, and exhibited typical corkscrewlike motility. After yolk-sac inoculation, the agent was highly lethal for chick embryos and was recovered in large quantity from several embryonic tissues and fluids. It could also be maintained in nonfertile eggs and in an enriched liquid medium. This previously undescribed spirochete could pass through Berkefeld N but not Seitz EK filters. It was relatively resistant to heat and to penicillin or sulfadiazine, but was markedly inhibited by streptomycin, broad-spectrum antibiotics, and homologous neutralizing antiserum. Of several species of animals tested for susceptibility to this spirochete, only the snowshoe hare gave evidence of infection.