Description of Oceanispirochaeta crateris sp. nov. and reclassification of Spirochaeta perfilievii as Thiospirochaeta perfilievii gen. nov., comb. nov.

A novel obligately anaerobic spirochete strain K2T was isolated from bottom marine sediments at Crater Bay of Yankicha Island (Kuril Islands, Russia). Strain K2T had helical shape and Gram-negatively stained. The optimal growth conditions were as follows: the optimum temperature was 28-30 °C with range 5-34 °C; optimal pH at 7.0-7.5 with range of 6.8-8.5; NaCl optimum at 3-3.5 % (w/v) and range of 1-7 % (w/v). Strain K2T was catalase- and oxidase-negative. Glucose fermentation products were acetate, lactate, ethanol, CO2, H2. The major fatty acids were C14 : 0, iso-C13 : 0, iso-C15:0, C14 : 0 DMA, iso-C15 : 0 DMA. The G+C content of genomic DNA was 43.2 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain K2T belonged to the genus Oceanispirochaeta of the family Spirochaetaceae. The 16S rRNA gene sequence similarity of strain K2T and O. litoralis DSM 2029T and O. sediminicola DSM 104770T was 96 and 94 %, respectively. Based on the results of our study, we propose the name Oceanispirochaeta crateris sp. nov.; type strain K2T (=DSM 16308T=VKM B-3266T). Also, the taxonomic status of Spirochaeta perfilevii was revised: 16S rRNA genes sequence showed less than 89 % similarity to nearest phylogenetic neighbours. Therefore, we proposed to separate this species into a novel genus Thiospirochaeta - T. perfilievii gen. nov., comb. nov.

Sphaerochaeta halotolerans sp. nov., a novel spherical halotolerant spirochete from a Russian heavy oil reservoir, emended description of the genus Sphaerochaeta, reclassification of Sphaerochaeta coccoides to a new genus Parasphaerochaeta gen. nov. as Parasphaerochaeta coccoides comb. nov. and proposal of Sphaerochaetaceae fam. nov.

Anaerobic, fermentative, halotolerant bacteria, strains 4-11T and 585, were isolated from production water of two low-temperature petroleum reservoirs (Russia) and were characterized by using a polyphasic approach. Cells of the strains were spherical, non-motile and 0.30-2.5 µm in diameter. Strain 4-11T grew optimally at 35 °C, pH 6.0 and 1.0-2.0% (w/v) NaCl. Both strains grew chemoorganotrophically with mono-, di- and trisaccharides. The major cellular fatty acids of both strains were C14:0, C16:0, C16:1 ω9 and C18:0 3-OH. Major polar lipids were glycolipids and phospholipids. The 16S rRNA gene sequences of the strains 4-11T and 585 had 99.9% similarity and were most closely related to the sequence of Sphaerochaeta associata GLS2T (96.9, and 97.0% similarity, respectively). The G+C content of the genomic DNA of strains 4-11T and 585 were 46.8 and 46.9%, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between the genomes of strain 4-11T and S. associata GLS2T were 73.0 and 16.9%, respectively. Results of phylogenomic metrics analysis of the genomes and 120 core proteins of strains 4-11T and 585 and their physiological and biochemical characteristics confirmed that the strains represented a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta halotolerans sp. nov. is proposed, with the type strain 4-11T (=VKM B-3269T=KCTC 15833T). Based on the results of phylogenetic analysis, Sphaerochaeta coccoides was reclassified as member of a new genus Parasphaerochaeta gen. nov., Parasphaerochaeta coccoides comb. nov. The genera Sphaerochaeta and Parasphaerochaeta form a separate clade, for which a novel family, Sphaerochaetaceae fam. nov., is proposed.

Rectinema subterraneum sp. nov, a chemotrophic spirochaete isolated from the deep terrestrial subsurface.

A novel, obligately anaerobic bacterium (strain SURF-ANA1T) was isolated from deep continental subsurface fluids at a depth of 1500 m below surface in the former Homestake Gold Mine (now Sanford Underground Research Facility, in Lead, South Dakota, USA). Cells of strain SURF-ANA1T were Gram-negative, helical, non-spore-forming and were 0.25-0.55×5.0-75.0 µm with a wavelength of 0.5-0.62 µm. Strain SURF-ANA1T grew at 15-50 °C (optimally at 40 °C), at pH 4.8-9.0 (pH 7.2) and in 1.0-40.0 g l-1 NaCl (10 g l-1 NaCl). The strain grew chemoheterotrophically with hydrogen or mono-, di- and polysaccharides as electron donors. The major cellular fatty acids in order of decreasing abundance (comprising >5% of total) were 10-methyl C16:0, iso-C15:0, C18:2 and C18:0 dimethyl acetal (DMA) and C20:0 methylene-nonadecanoic acid. Phylogenetic analysis based on the 16S rRNA gene sequence of strain SURF-ANA1T indicated a closest relationship with the recently characterized Rectinema cohabitans (99%). Despite high sequence identity, because of its distinct physiology, morphology and fatty acid profile, strain SURF-ANA1T is considered to represent a novel species within the genus Rectinema, for which the name Rectinema subterraneum sp. nov. is proposed. To our knowledge, this is the first report of an isolate within the phylum Spirochaetes from the deep (>100 m) terrestrial subsurface. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genomic sequences of strain SURF-ANA1T are KU359248 and GCF 009768935.1, respectively. The type strain of Rectinema subterraneum is SURF-ANA1T (=ATCC TSD-67=JCM 32656).

Occurrence of Borrelia and Borreliella species in Ixodes ricinus collected from roe deer in northwestern Spain.

Ixodes ricinus, comprising the predominant tick species in Europe, can transmit important human pathogens, including Borreliella spp., the causal agent of Lyme borreliosis. One hundred and seventy five roe deer hunted in two areas (plateau and mountain) of Galicia (northwest Spain) were examined for the presence of ticks; all roe deer were infested by I. ricinus. Nymphs (n = 1000), males (n = 1449) and females (n = 1000) of I. ricinus were analysed in pools of up to 10 ticks to detect both Borreliella and Borrelia DNA. The average number of I. ricinus per roe deer was similar in both areas, regardless of the life stage; although the percentage of Borreliella and Borrelia positive pools was higher in ticks collected from roe deer hunted in the plateau area, no significant differences were detected. Sequence analysis at the flagellin gene allowed the identification of four Borreliella species (Borreliella afzelii, Borreliella garinii, Borreliella lusitaniae and Borreliella valaisiana) and Borrelia miyamotoi in adult males; only B. valaisiana and B. miyamotoi were detected in nymphs and all females were negative. All Borreliella and Borrelia species found in roe deer were previously identified in questing I. ricinus collected in the same study area, although the prevalence was lower in the present study. The analysis of male I. ricinus ticks collected from roe deer gives a good estimation of Borreliella diversity in questing ticks.

Pleomorphochaeta naphthae sp. nov., a new anaerobic fermentative bacterium isolated from an oil field.

A novel anaerobic fermentative bacterium, strain SEBR 4209T, was isolated from a water sample of a Congolese oil field. Strain SEBR 4209T is phylogenetically related to the genus Pleomorphochaeta, in the family Spirochaetaceae. Its closest relatives are Pleomorphochaeta caudata SEBR 4223T (94.5 % 16S rRNA gene sequence similarity) and Pleomorphochaeta multiformis MO-SPC2T (94.3 % similarity). Like the other members of this genus, cells have a pleomorphic morphology, in particular an annular shape and long stalks. Optimal growth was observed at 37 °C, at pH between 6.8 and 7.0, and with 40 g l-1 NaCl. This strain was only able to grow by fermentation of carbohydrates. The fermentation products from glucose utilization were acetate, ethanol, CO2 and H2. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c. The major polar lipids were phosphoglycolipids, phospholipids and glycolipids. The G+C content of the DNA was 29.6 mol%. Based on phenotypic characteristics and phylogenetic traits, strain SEBR 4209T is considered to represent a novel species of the genus Pleomorphochaeta, for which the name Pleomorphochaetanaphthae sp. nov. is proposed. The type strain is SEBR 4209T (=DSM 104684T=JCM 31871T).

Characterisation of a newly isolated member of a candidatus lineage, Marispirochaeta aestuarii gen. nov., sp. nov.

Metagenome analysis of coastal marine habitats of Gujarat, India indicated the presence of twelve novel putative lineages of spirochaetes. Out of which a strain designated JC444T representing a novel putative lineage seven was isolated and characterized based on a polyphasic taxonomic approach. Strain JC444T was helical, Gram-stain-negative, obligate anaerobe, catalase and oxidase negative. Strain JC444T was able to grow at 15-45 °C (optimum at 30-35 °C), pH 6.5-8.6 (optimum at 7.5-8.0) and 0.6-5 % (optimum at 1.5-2.0 %) of NaCl concentration. The major end products of glucose fermentation were acetate, formate, hydrogen and carbon dioxide. C14 : 0, iso-C15 : 0, C16 : 0, C18 : 0, iso-C15 : 1H/C13 : 03OH (summed feature 1), iso-C13 : 0, anteiso-C15 : 0 and iso-C17 : 0 were present as fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unidentified lipids (L1-4) were the polar lipids. G+C mol% of strain JC444T was 53.6 %. 16S rRNA gene sequence comparisons indicated that strain JC444T represents a member of the family Spirochaetaceae in the order Spirochaetales. Strain JC444T has a sequence similarity of 97.1 % with 'Candidatus Marispirochaeta associata' JC231 and <90.1 % with other members of the family Spirochaetaceae. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain JC444T as a representative of a new genus and species in the family Spirochaetaceae, for which the name Marispirochaeta aestuarii gen. nov., sp. nov. is proposed. Type strain is JC444T (=KCTC 15554T=DSM 103365T).

Description of Oceanispirochaeta sediminicola gen. nov., sp. nov., an obligately anaerobic bacterium isolated from coastal marine sediments, and reclassification of Spirochaeta litoralis as Oceanispirochaeta litoralis comb. nov.

An obligately anaerobic spirochaete (strain SY2T) was isolated from coastal marine sediments of Tongyeong-Si, South Korea. Strain SY2T was helical-shaped and Gram-stain-negative. Strain SY2T was able to grow at 10-40 °C (optima, 25-30 °C), pH 6.3-8.8 (optima, pH 7.0-8.0) and with 1-7 % (optimum, 2-3 %) NaCl concentration. Strain SY2T was negative for catalase and oxidase activity. The major end-products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. C14 : 0, C16 : 0, iso-C15 : 0, iso-C14 : 0 3-OH, iso-C15 : 1 H/C13 : 0 3-OH and iso-C17 : 1ω9c were predominant fatty acids (>5 %) with minor amounts (<5 %) of C18 : 0, iso-C13 : 0, iso-C17 : 0, iso-C17 : 1/anteiso-C17 : 1 B and C16 : 1ω6c/C16 : 1ω7c. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were major polar lipids. The genomic DNA G+C content was 53.5 mol%. 16S rRNA gene sequence comparisons indicated that strain SY2T represents a member of the family Spirochaetaceae in the phylum Spirochaetes. Strain SY2T has a sequence similarity of 95.1 % with Spirochaeta litoralis R1T and <90.1 % with other members of the genus Spirochaeta. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain SY2T as a representative of a novel genus and species in the family Spirochaetaceae, for which the name Oceanispirochaeta sediminicola gen. nov., sp. nov. is proposed. The type strain is SY2T (=KEMB 3001-381T=DSM 104770T=KCTC 15593T). Reclassification of Spirochaeta litoralis as Oceanispirochaeta litoralis comb. nov. is also proposed based on polyphasic taxonomic analyses.

The microbiome of the octocoral Lobophytum pauciflorum: minor differences between sexes and resilience to short-term stress.

Bacteria associated with marine invertebrates are thought to have a range of important roles that benefit the host including production of compounds that may exclude pathogenic microorganisms and recycling of essential nutrients. This study characterised the microbiome of a gonochoric octocoral, Lobophytum pauciflorum, and investigated whether either sex or environmental stresses influenced the diversity of the associated microbiome through amplicon profiling of the bacterial 16S rRNA gene. Sequences affiliated to Spirochaetaceae and Endozoicimonaceae dominated the microbiome of L. pauciflorum, representing 43% and 21% of the community, respectively. Among the dominant class affiliations, no sex-specific differences were detected, though unassigned sequences were at a 2-fold higher relative abundance in samples from female individuals than from males. These potentially novel sequences contributed to observed differences between sexes as detected by a multivariate analysis at the OTU level. Exposing L. pauciflorum fragments to increased temperature (31°C), decreased pH (7.9) or both stressors simultaneously for 12 days did not significantly alter the microbial community, indicating that the soft coral microbiome is relatively resilient to short-term environmental stress.

Pleomorphochaeta caudata gen. nov., sp. nov., an anaerobic bacterium isolated from an offshore oil well, reclassification of Sphaerochaeta multiformis MO-SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov. as the type strain of this novel genus and emended description of the genus Sphaerochaeta.

A strictly anaerobic Gram-stain-negative bacterium, designated strain SEBR 4223T, was isolated from the production water of an offshore Congolese oil field. Cells were non-motile, pleomorphic and had spherical, annular or budding shapes, often exhibiting long stalks. Strain SEBR 4223T grew on a range of carbohydrates, optimally at 37 °C and pH 7, in a medium containing 40 g l-1 NaCl. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c and the major polar lipids were phosphoglycolipids, phospholipids, glycolipids and diphosphatidylglycerol. The G+C content of the DNA was 28.7 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain SEBR 4223T and Sphaerochaeta multiformis MO-SPC2T formed a cluster with similarity to other species of the genus Sphaerochaeta of of less than 86 %. On the basis of the phenotypic characteristics and taxonomic analyses, we propose a novel genus, Pleomorphochaeta gen. nov., to accommodate the novel species Pleomorphochaeta caudata sp. nov., with SEBR 4223T (=DSM 103077T=JCM 31 475T) as the type strain. We also propose the reclassification of Sphaerochaeta multiformis MO SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov., the type strain of this novel genus and emend description of the genus Sphaerochaeta.

Sphaerochaeta associata sp. nov., a spherical spirochaete isolated from cultures of Methanosarcina mazei JL01.

An anaerobic, saccharolytic bacterial strain designated GLS2T was isolated from aggregates of the psychrotolerant archaeon Methanosarcina mazei strain JL01 isolated from arctic permafrost. Bacterial cells were non-motile, spherical, ovoid and annular with diameter 0.2-4 µm. They were chemoorganoheterotrophs using a wide range of mono-, di- and trisaccharides as carbon and energy sources. The novel isolate required yeast extract and vitamins for growth. The bacteria exhibited resistance to a number of ß-lactam antibiotics, rifampicin, streptomycin and vancomycin. Optimum growth was observed between 30 and 34 °C, at pH 6.8-7.5 and with 1-2 g NaCl l- 1. Isolate GLS2T was a strict anaerobe but it tolerated oxygen exposure. On the basis of 16S rRNA gene sequence similarity, strain GLS2T was shown to belong to the genus Sphaerochaeta within the family Spirochaetaceae. Its closest relatives were Sphaerochaeta globosa BuddyT (99.3 % 16S rRNA gene sequence similarity) and Sphaerochaeta pleomorpha GrapesT (95.4 % similarity). The G+C content of DNA was 47.2 mol%. The level of DNA-DNA hybridization between strains GLS2T and BuddyT was 34.7 ± 8.8 %. Major polar lipids were phosphoglycolipids, phospholipids and glycolipids; major fatty acids were C14 : 0, C16 : 0, C16 : 0 3-OH, C16 : 0 dimethyl acetal (DMA), C16 : 1n8 and C16 : 1 DMA; respiratory quinones were not detected. The results of DNA-DNA hybridization, physiological and biochemical tests demonstrated genotypic and phenotypic differentiation of strain GLS2T from the four species of the genus Sphaerochaeta with validly published names that allowed its separation into a new lineage at the species level. Strain GLS2T therefore represents a novel species, for which the name Sphaerochaeta associata sp. nov. is proposed, with the type strain GLS2T ( = DSM 26261T = VKM B-2742T).

Sphaerochaeta multiformis sp. nov., an anaerobic, psychrophilic bacterium isolated from subseafloor sediment, and emended description of the genus Sphaerochaeta.

An anaerobic, psychrophilic bacterium, strain MO-SPC2(T), was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediments collected from off the Shimokita Peninsula of Japan in the north-western Pacific Ocean. Cells were pleomorphic: spherical, annular, curved rod, helical and coccoid cell morphologies were observed. Motility only occurred in helical cells. Strain MO-SPC2(T) grew at 0-17 °C (optimally at 9 °C), at pH 6.0-8.0 (optimally at pH 6.8-7.2) and in 20-40 g NaCl l(-1) (optimally at 20-30 NaCl l(-1)). The strain grew chemo-organotrophically with mono-, di- and polysaccharides. The major end products of glucose fermentation were acetate, ethanol, hydrogen and carbon dioxide. The abundant polar lipids of strain MO-SPC2(T) were phosphatidylglycolipids, phospholipids and glycolipids. The major cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9. Isoprenoid quinones were not detected. The G+C content of the DNA was 32.3 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-SPC2(T) was affiliated with the genus Sphaerochaeta within the phylum Spirochaetes, and its closest relatives were Sphaerochaeta pleomorpha Grapes(T) (88.4 % sequence identity), Sphaerochaeta globosa Buddy(T) (86.7 %) and Sphaerochaeta coccoides SPN1(T) (85.4 %). Based on phenotypic characteristics and phylogenetic traits, strain MO-SPC2(T) is considered to represent a novel species of the genus Sphaerochaeta, for which the name Sphaerochaeta multiformis sp. nov. is proposed. The type strain is MO-SPC2(T) ( = JCM 17281(T) = DSM 23952(T)). An emended description of the genus Sphaerochaeta is also proposed.

Selected phenotypic features of BR91, a unique spirochaetal strain isolated from the Culex pipiens mosquito.

Growth temperature range, resistance to selective antibiotics, activities of 23 enzymes, protein fingerprints and fatty acids composition of the spirochaetal strain BR91, isolated from the Culex pipiens mosquito, were tested. The spirochaetes were grown in BSK-H Complete liquid medium. The optimal in vitro growth temperature of the strain was 33 °C. Strain BR91 was sensitive to trimethoprim, nalidixic acid, 5-fluorouracil, and tolerated phosphomycin. The strain produced acid and alkaline phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and α-fucosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay revealed several major proteins in the size range of 13-16 kDa, 22-30 kDa and 37-131 kDa. Fatty acid methyl ester (FAME) analysis showed that C14:0, C16:0, C18:1 ω9c and summed feature 5 (C18:2 ω6,9c and/or C18:0 anteiso) are major fatty acids. This study highlights certain phenotypic differences between strain BR91 and the Lyme disease spirochaete Borrelia burgdorferi, and supports the hypothesis that strain BR91 represents a unique taxonomical entity in a system of spirochaetal species.

Freqüência de anticorpos homólogos anti-Borrelia burgdorferi em eqüinos na mesorregião metropolitana de Belém, Estado do Pará / Occurrence of homologous anti-Borrelia burgdorferi antibodies in equines in the metropolitan mesorregion of Belém, State of Para, Brazil

Espiroquetas transmitidas por carrapatos são microrganismos de ampla distribuição geográfica e acometem animais silvestres, domésticos e seres humanos. Procedeu-se a análise sorológica de 300 soros de eqüinos onde 58 animais eram do município Ananideua, 61 eram de Belém, 131 de Castanhal e 50 eram do município de Santa Izabel do Pará para Borrelia burgdorferi através do teste ELISA indireto. Não foram observadas diferenças significativas (P < 0,05) entre os municípios, nem quanto à raça, sexo e função dos animais. Um total de 80 (26,7 por cento) animais foram positivos para B. burgdorferi com os títulos de 1:800, 72 (90 por cento) eqüinos; 1:1.600, 6 (7,5 por cento) eqüinos; e 1:3.200, 2 (2,5 por cento) eqüinos. Os resultados observados foram similares aos descritos nos EUA, onde foram relatadas freqüências de soropositivos variando entre 7 e 75 por cento em eqüinos assintomáticos. A presença de anticorpos homólogos contra B. burgdorferi em eqüinos na mesorregião metropolitana de Belém é indicativo da ampla distribuição do agente e da possibilidade de ocorrerem casos humanos deste agente na região.

Spirochaetes transmitted by ticks are microorganisms of worldwide distribution, which infect wild, domestic animals and human beings. A total of 300 equine sera from four municipalities: Ananideua (58), Belém (61), Castanhal (131), and Santa Izabel do Pará (50), were evaluated for Borrelia burgdorferi by an Elisa test. There were no significant differences (P<0.05) among municipalities, breed, sex or husbandry. A total of 80 (26.7 percent) horses were B. burgdorferi positive with titles of 1:800, 72 (90 percent) horses, 1:1.600, 6 (7.5 percent) horses, and 1:3.200, 2 (2.5 percent) horses. The results were similar to those in the USA, where related frequencies ranged from 7 to 75 percent in asymptomatic seropositive horses. The presence of anti-B.burgdorferi homologous antibodies in horses from four municipalities in the metropolitan mesorregion of Belém suggests the possibility of occurrence of human cases in the region.

A novel enteropathogenic, strongly haemolytic spirochaete isolated from pig and mallard, provisionally designated 'Brachyspira suanatina' sp. nov.

Atypical, strongly haemolytic porcine isolates of intestinal spirochaetes differing genetically from Brachyspira hyodysenteriae were identified and characterized. The isolates were subjected to culture and biochemical tests, antimicrobial susceptibility testing and molecular analyses. None of four species-specific polymerase chain reaction systems targeting genes of B. hyodysenteriae gave a positive reaction. All the atypical porcine isolates were identical in their partial 16S rRNA and nox gene sequences with a previously described isolate from a mallard (Anas platyrhynchos), and differed only slightly from another mallard isolate. All these isolates were distinctly different from all currently recognized Brachyspira species. A challenge study was carried out using recently weaned pigs. Clinical signs and macroscopic changes consistent with swine dysentery were seen both in pigs given the atypical porcine isolate and in control pigs given the reference strain of B. hyodysenteriae (B204(R)). Pigs given the genetically similar isolate from a mallard became colonized and diarrhoea was observed. This is the first study indicating that Brachyspira isolates from mallard can infect pigs and induce diarrhoea. We propose that this atypical spirochaete genotype should be regarded as a new species within the genus Brachyspira, and be provisionally designated 'Brachyspira suanatina' sp. nov.

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test.

There is no ring test for quality assessment available in Europe for diagnostics and antimicrobial susceptibility testing of the fastidious, anaerobic bacteria of the genus Brachyspira. Therefore, an international ring test for Brachyspira spp. was performed once a year during 2002-2004. Two sets of coded samples were prepared and distributed on each occasion. One set comprised six swabs dipped in pig faeces spiked with Brachyspira spp. intended for diagnostics. The other set comprised two pure strains intended only for susceptibility testing. All methods used were in-house methods. The species used were Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira innocens, Brachyspira murdochii and Brachyspira intermedia. In most cases, the correct Brachyspira spp. were detected. However, the results showed that Brachyspira spp. could be difficult to identify, especially if two Brachyspira spp. were mixed or if the concentration of Brachyspira in faeces was low. Additionally, some laboratories reported Brachyspira growth in control samples that were not seeded with any spirochaetes. The lowest detection level was 10(2) bacteria/ml faeces for both B. hyodysenteriae and B. pilosicoli. The susceptibility tests performed showed that disc diffusion was not recommendable for Brachyspira spp. Extended antimicrobial dilution series gave most congruent results. The diversity of the results highlights the importance of ring tests for a high quality of diagnostics and antimicrobial susceptibility tests for Brachyspira spp. This is the first ring test described for Brachyspira spp.

Occurrence of Lawsonia Intracellularis and Brachyspira spp. infection in swine suffering from diarrhoea.

Principal aim of this study was to examine fecal samples from pigs suffering from diarrhea for the presence of Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli. The molecular techniques such as PCR and nested PCR were employed to detect the presence of p78 fragment of genomic DNA specific for Lawsonia intracellularis as well as fragment of tlyA gene specific for Brachyspira hyodysenteriae and 16S rDNA gene of Brachyspira pilosicoli. We assumed that about 25% of pigs were infected with Lawsonia intracellularis, about 10% with Brachyspira hyodysenteriae and only 0,8% with Brachyspira pilosicoli. In about 3% mixed infection with L. intracellularis and B. hyodysenteriae was observed. Results were comparable in herds that differed in quantity, breeding technology, hygienic standards and preventive treatment with different chemotherapeutics.

[Human intestinal spirochetosis]. / Intestinální spirochetóza cloveka.

A characteristic feature of human intestinal spirochetosis (IS) is the colonization of the mucosa of the large intestine with intestinal spirochetes of the genus Brachyspira. The joining of the brachyspirae with the apical cellular membrane of enterocytes resembles in histological slides a false brush border of the intestinal mucosa. Various symptoms related to the involvement of the large gut were found with invasive IS. From the cultures of these cases were isolated Brachyspira aalborgii and B. pilosicoli. The frequency of the incidence of brachyspirae depended on the socio-economic living conditions of people. Colonization of the mucosa of the large gut was found more often in human populations in the developing countries; it was fairly rare in countries with high hygienic standards. An exception were men of homosexual orienation and patients presenting with a HIV infection. Isolation of brachyspirae from the faeces and biopsy of the mucosa of the large gut are fairly demanding jobs, especially with B. aalborgii. Most documented IS cases of this aetiology were diagnosed using immunohistochemical methods and amplification of the genus-specific region of the gene 16S rRNA. Isolation of B. pilosicoli tends to be simpler, it requires anaerobic incubation on selective blood agars for a period of 3-6 days at 37 degrees C. When manual haemoculture systems were used, patients in a critical state presented a translocation of brachyspirae into blood circulation, while automatic systems don't necessarily diagnose spirochetaemia. In the management of described cases of invasive IS particularly successful proved metronidazole and beta-lactam antibiotics. In isolated B. pilosicoli, in vitro tests confirmed sensitivity to metronidazole, ceftriaxone, meropenem, tetracycline, moxifloxacine and chloramphenicol. A varying frequency of resistance was found with clindamycin and amoxicillin, which how ever was efficacious in combination with clavulanic acid.