Strains Of Spirochaetes Research Articles

Brachyspira catarrhinii sp. nov., an anaerobic intestinal spirochaete isolated from vervet monkeys may have been misidentified as Brachyspira aalborgi in previous studies

To date nine species of anaerobic intestinal spirochaetes have been validly assigned to the genus Brachyspira. These include both pathogenic and non-pathogenic species. In the current study a genomic analysis of a novel spirochaete isolate was undertaken to determine whether it is a distinct species that previously has been misidentified as Brachyspira aalborgi. The genome of spirochaete strain Z12 isolated from the faeces of a vervet monkey was sequenced and compared to the genomes of the type strains of the nine assigned Brachyspira species. Genome to Genome Distance (GGD) values and Average Nucleotide Identity (ANI) values were determined. Single nucleotide polymorphisms (SNP) were used to create a phylogenetic tree to assess relatedness. The 16S rRNA gene sequences of the strains were aligned and the similarity amongst the Brachyspira species was recorded. Multilocus sequence typing (MLST) using five loci was conducted on Z12 and results compared with those for other Brachyspira isolates. Assembly of the Z12 sequences revealed a 2,629,108 bp genome with an average G + C content of 31.3%. The GGD, ANI, 16S rRNA gene sequence comparisons and the MLST results all indicated that Z12 represents a distinct species within the genus Brachyspira, with its nearest neighbour being B. aalborgi. Spirochaete strain Z12T was assigned as the type strain of a new species, Brachyspira catarrhinii sp. nov. The diagnostic PCR currently in use to detect B. aalborgi cross-reacts with Z12, but RFLP analysis of PCR product can be used to distinguish the two species. Previous reports of non-human primates being colonised by B. aalborgi based on PCR results may have been incorrect. The development of an improved diagnostic method will allow future studies on the distribution and possible clinical significance of these two anaerobic spirochaete species.

Typhlocolitis associated with spirochaetes in duck flocks

The aetiology of increased mortality observed in two breeder duck flocks (Flock A consisting of 3500 laying ducks and Flock B comprising 4300 laying ducks) during the first egg-laying season was studied. In Flocks A and B, 773 ducks and 715 ducks (18.4% and 16.6%) died within a 24-week and a 20-week period, respectively. Death was preceded by clinical signs including movement difficulties, lack of appetite and depression lasting for 1 to 2 days. Diarrhoea was not observed. On gross pathological examination, the ducks were found to have haemorrhagic to fibrinonecrotic typhlocolitis, renal degeneration accompanied by fibrosis and mineralization, hepatic and splenic amyloidosis, and swelling of some of the metatarsal and phalangeal joints. Histopathological and immunohistochemical examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. On the basis of their cultural and biochemical properties and polymerase chain reaction sequencing analysis, four out of seven spirochaete strains isolated from the ducks (Flock A) by culture on special media under anaerobic conditions were identified as Brachyspira hyodysenteriae, and five out of eight strains (Flock B) were identified as Brachyspira pilosicoli. This is the first report on the isolation of B. hyodysenteriae and B. pilosicoli from laying ducks affected by fibrinonecrotic typhlocolitis.

Multilocus sequence analysis of Borrelia bissettii strains from North America reveals a new Borrelia species, Borrelia kurtenbachii

Using multilocus sequence analysis (MLSA), we investigated the phylogenetic relationship of spirochaete strains from North America previously assigned to the genospecies Borrelia bissettii. We amplified internal fragments of 8 housekeeping genes ( clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) located on the main linear chromosome by polymerase chain reaction. Phylogenetic analysis of concatenated sequences of the 8 loci showed that the B. bissettii clade consisted of 4 closely related clusters which included strains from California (including the type strain DN127-Cl9-2/p7) and Colorado that were isolated from Ixodes pacificus, I. spinipalpis, or infected reservoir hosts. Several strains isolated from I. scapularis clustered distantly from B. bissettii. Genetic distance analyses confirmed that these strains are more distant to B. bissettii than B. carolinensis, a recently described Borrelia species, which suggests that they constitute a new Borrelia genospecies. We propose that it be named Borrelia kurtenbachii sp. nov. in honour of the late Klaus Kurtenbach. The data suggest that ecological differences between B. bissettii and the new Borrelia genospecies reflect different transmission cycles. In view of these findings, the distinct vertebrate host-tick vector associations and the distributions of B. bissettii and B. kurtenbachii require further investigation.

Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens

Anaerobic intestinal spirochaetes of the genus Brachyspira include several important pathogenic species, particularly those infecting pigs and chickens. In this study a multiplex-quantitative polymerase chain reaction (M-qPCR) assay was developed based on amplification of a 198 base pair portion of the NADH oxidase gene, using TaqMan probes for detecting and quantifying the three main pathogenic species, B. hyodysenteriae, B. pilosicoli and B. intermedia. The specificity of the assay was validated using 130 spirochaete strains belonging to members of the seven officially named and two provisionally named Brachyspira species. The detection limit for all three targeted species was 1–10 viable cells and 10 fg DNA per reaction. Further detection limit testing was conducted on porcine and chicken faecal specimens that were spiked with spirochaete cells before DNA extraction. The assay could detect 10 2 to 10 3 cells per 0.2 g of sample, giving an improved detection threshold compared to standard PCRs. The M-qPCR was further developed by incorporating a novel internal control (IC) that employed host cells as template DNA. This adaptation allowed monitoring of the quality of the extracted DNA and ensured that there was no inhibition of the PCR reaction. Use of the IC further improved the detection limits of the assay and increased confidence in being able to detect low numbers of pathogens in faecal samples. Taken together, the results indicate that the new M-qPCR assay is a valuable tool for detecting and quantifying low numbers of pathogenic intestinal spirochaetes in the faeces of pigs and chickens, and potentially other species.

Identification of genes associated with prophage-like gene transfer agents in the pathogenic intestinal spirochaetes Brachyspira hyodysenteriae, Brachyspira pilosicoli and Brachyspira intermedia

VSH-1 is an unusual prophage-like gene transfer agent (GTA) that has been described in the intestinal spirochaete Brachyspira hyodysenteriae. The GTA does not self-propagate, but it assembles into a virus-like particle and transfers random 7.5 kb fragments of host DNA to other B. hyodysenteriae cells. To date the GTA VSH-1 has only been analysed in B. hyodysenteriae strain B204, in which 11 late function genes encoding prophage capsid, tail and lysis elements have been described. The aim of the current study was to look for these 11 genes in the near-complete genomes of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. intermedia HB60. All 11 genes were found in the three new strains. The GTA genes in WA1 and 95/1000 were contiguous, whilst some of those in HB60 were not—although in all three strains some gene rearrangements were present. A new predicted open reading frame with potential functional importance was found in a consistent position associated with all four GTAs, located between the genes for head protein Hvp24 and tail protein Hvp53, overlapping with the hvp24 sequence. Differences in the nucleotide and predicted amino acid sequences of the GTA genes in the spirochaete strains were consistent with the overall genetic distances between the strains. Hence the GTAs in the two B. hyodysenteriae strains were considered to be strain specific variants, and were designated GTA/Bh-B204 and GTA/Bh-WA1 respectively. The GTAs in the strains of B. intermedia and B. pilosicoli were designated GTA/Bint-HB60 and GTA/Bp-95/1000 respectively. Further work is required to determine the extent to which these GTAs can transfer host genes between different Brachyspira species and strains.

Identification and characterization of 31 isolates of Borrelia burgdorferi (Spirochaetales, Spirochaetaceae) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

AbstractBorrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes ricinus (L., 1758) ticks. The aim of our study was to identify spirochaete isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their characterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infraorder Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.

Isolation and molecular characterization of a Brazilian strain of Borrelia anserina, the agent of fowl spirochaetosis

Borrelia anserina the agent of fowl spirochaetosis, has a worldwide distribution, where it is transmitted by Argas spp. ticks. The present study reports the first molecular characterization and in vitro isolation of an avian spirochaete strain from Brazil, presumably identified as B. anserina originated from naturally infected Argas miniatus ticks. DNA fragments of the rrs and flab genes were amplified by PCR and sequenced to determine phylogenetic similarities. The resulting sequences were 99.8% (483 of 484) and 98.7% (754 of 764) similar to GenBank corresponding sequences of B. anserina rrs and flaB genes, respectively. By neighbor-joining phylogenetic analysis, the flaB sequence of the Brazilian strain clustered in a monophyletic group with the sequence of B. anserina under 100% bootstrap support. The isolate was successfully isolated in BSK medium, with seven passages performed. The spirochaete strain isolated in the present study was genetically identified as B. anserina labeled as strain PL.

Typhlocolitis associated with spirochaetes in goose flocks

The role of Brachyspira bacteria in the aetiology of increased mortality observed in two breeder goose flocks (Flock A consisting of 1,500 and Flock B comprising 4,500 laying geese) at the end of the first egg-laying season, in the period of moulting, was studied. In Flock A 415 geese (28%) died during an 8-week period while in Flock B 834 geese (18%) died during a 12-week period. On gross pathological examination, the geese were found to have haemorrhagic-to-necrotic inflammation of the large intestine (colon and rectum) and fibrinonecrotic typhlitis accompanied by severe degeneration. Often, fibrosis of the kidneys, and in five of the geese secondary visceral urate deposition ("visceral gout") was also observed. Histopathological examination consistently demonstrated spirochaetes in the mucous membrane of the affected large intestine. This was confirmed by the results of immunohistochemical and electron microscopic examination. In addition, Trichomonas stages were also detected from the large intestine of 11 geese. On the basis of their cultural and biochemical properties, and PCR sequencing analysis, eight out of the nine spirochaete strains isolated from the geese by culture on special media under anaerobic conditions were identified as Brachyspira alvinipulli. This is the first report on the isolation of B. alvinipulli from laying geese affected with fibrinonecrotic typhlocolitis.

Survival of intestinal spirochaete strains from chickens in the presence of disinfectants and in faeces held at different temperatures

This study aimed to evaluate the efficacy of some commonly used disinfectants in inactivating the pathogenic avian intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli, and to examine spirochaete survival in chicken caecal faeces held at 4°C, 25°C or 37°C. Six disinfectants were evaluated at their recommended working concentrations: alkaline salts, quaternary ammonium, iodine as an iodophor, chlorine from a chlorine-release agent, glutaraldehyde and hydrogen peroxide. All but alkaline salts inactivated two different concentrations of both spirochaete species in less than 1 min in the presence of organic matter. Both spirochaete species at three different cell concentrations survived in caecal faeces at 37°C for between 2 and 17 h. B. intermedia tended to survive for longer than B. pilosicoli, but the maximum survival time for both species at 4°C was only 72 to 84 h. Hence, avian intestinal spirochaetes are rapidly inactivated by several common disinfectants, and their survival time in chicken caecal faeces is much less than has been reported for porcine intestinal spirochaetes in porcine faeces. It should be relatively easy to break the cycle of infection between batches of laying birds by resting sheds for a few days, and by using disinfectants on any residual faecal matter.

Differentiation of Serpulina species by NADH oxidase gene ( nox) sequence comparisons and nox-based polymerase chain reaction tests

The NADH oxidase genes ( nox) of 18 strains of intestinal spirochaetes were partially sequenced over 1246 bases. Strains examined included 17 representatives from six species of the genus Serpulina, and the type strain 513A T of the human intestinal spirochaete Brachyspira aalborgi. Sequences were aligned and used to investigate phylogenetic relationships between the organisms. Nox sequence identities between strains within the genus Serpulina were within the range 86.3–100%, whilst the nox gene of B. aalborgi shared between 78.8–83.0% sequence identity with the nox sequences of the various Serpulina strains. A phenogram produced based on sequence dissimilarities was in good agreement with the current classification of species in the genus Serpulina, although an atypical strongly beta-haemolytic porcine strain (P280/1), previously thought to be S. innocens, appeared distinct from other members of this species. Primer pairs were developed from the nox sequence alignments for use in polymerase chain reaction (PCR) identification of the pathogenic species S. hyodysenteriae (NOX1), S. intermedia (NOX2), and S. pilosicoli (NOX3), and for the combined non-pathogenic species S. innocens and S. murdochii (NOX4). The PCRs were optimised using 80 strains representing all currently described species in the genus Serpulina, as well as the type strain of B. aalborgi. Tests NOX1 and NOX4 specifically amplified DNA from all members of their respective target species, whilst tests NOX2 and NOX3 were less sensitive. NOX2 amplified DNA from all 10 strains of S. intermedia from pigs but from only 4 of 10 strains from chickens, whilst NOX3 amplified DNA from only 18 of 21 S. pilosicoli strains, even at low stringency. Tests NOX1 and NOX4 should prove useful in veterinary diagnostic laboratories, whilst NOX2 and NOX3 require further refinement.

Serpulina alvinipulli sp. nov., a new Serpulina species that is enteropathogenic for chickens.

Strain C1T is an anaerobic spirochaete that causes intestinal disease in chickens. Multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons have indicated that this spirochaete is a Serpulina strain. In these investigations, various phenotypic and genomic properties useful for establishing a taxonomic identity for strain C1T were studied. As determined by electron microscopy, cells of the spirochaete measured 8-11 x 0.22-0.34 mum and had a typical spirochaete ultrastructure. Each cell had 22-30 flagella. C1T cells formed weakly beta-haemolytic colonies on trypticase soy agar plates containing 5% bovine blood. The spirochaete reached maximum population densities of 10(9) cells ml-1 with a 2-4 h population doubling time in brain heart infusion broth containing 10% calf serum (BHIS broth). C1T cultures in BHIS broth were positive in tests for hippurate hydrolysis and negative for indole production. Glucosamine, N-acetyglucosamine, glucose, fructose, maltose and mannose were growth substrates for the spirochaete in heart infusion broth containing 7% calf serum (HS broth). During growth in HS broth beneath an O2/N2 (1:99) atmosphere, cells of the spirochaete consumed O2 and glucose and produced H2, CO2, acetate, butyrate and ethanol. Strain C1T DNA had a G+C content of 24.6 mol%. Based on DNA-DNA hybridization analyses, the DNA of strain C1T exhibited 24-39% relative reassociation with DNA of Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Serpulina murdochii and Serpulina intermedia. These results indicate that chicken spirochaete strain C1T has many phenotypic properties common to Serpulina species and, based on DNA hybridization analysis, represents a unique Serpulina species. For this new species the name Serpulina alvinipulli is proposed, for which the type strain is C1T (= ATCC 51933T).

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78 T, S. innocens strain B256 T, Brachyspira aalborgi strain 513A T, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes ( genetic distances between groups = 0.6–0.9). Chicken spirochaete strain C1 and B. aalborgi 513A T represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3–99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/ Serpulina species.

A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans

A monoclonal antibody (mAb) designed BJL/AC1 was prepared against the cell envelope of an intestinal spirochaete (strain 3295) that was isolated from a pig with intestinal spirochaetosis. The mAb reacted with a band of approximately 29 kDa in cell envelope preparations from 13 porcine and 11 human spirochaetes isolated from cases of intestinal spirochaetosis, but did not react with preparations made from a range of other intestinal spirochaetes. Immunogold labelling demonstrated that the reactive epitope was located on the cell envelope of the strains causing intestinal spirochaetosis. The mAb was used in an indirect immunofluorescence test to detect spirochaetes in the faeces of pigs with experimentally induced intestinal spirochaetosis. The mAb should prove to be a useful reagent for detection and identification of spirochaetes that are specifically associated with intestinal spirochaetosis.

Distribution of the smpA gene from Serpulina hyodysenteriae among intestinal spirochaetes.

Forty intestinal spirochaete strains were investigated for nucleotide sequences related to the smpA locus from Serpulina hyodysenteriae by Southern hybridization of chromosomal DNA using the smpA locus from S. hyodysenteriae strain P18A as a probe and by PCR using primers internal to the smpA gene. The intensity of the hybridization signal at high stringency and positive PCR results suggested that 12 S. hyodysenteriae strains possessed a similar nucleotide sequence. PCR was negative for another 12 S. hyodysenteriae strains and the hybridization signal obtained from 11 of these was weak and one was negative. All S. hyodysenteriae strains hybridized under low stringency conditions. These results indicated that there is variation among the smpA loci of S. hyodysenteriae strains. Among seven strains of S. innocens, and the proposed species 'S. intermedius' and 'S. murdochii', hybridization was weak and no PCR products were obtained, suggesting that these species have sequences related to, but divergent from, the smpA sequences of strains of S. hyodysenteriae. Both gene probe hybridization and PCR analysis of nine strains of the proposed new genus 'Anguillina', including isolates from pigs and humans, gave negative results.

Survey of Intestinal Spirochaetes for NADH Oxidase by Gene Probe and by Enzyme Assay

NADH oxidase activity has been found in a number of host-associated bacterial species by previous investigators. NADH oxidase may play an essential role in the colonisation of the mammalian intestinal tract by diverse bacterial species. In this study, 45 strains of intestinal spirochaetes were screened for NADH oxidase by enzyme assay and by using an oligodeoxynucleotide probe (5'-ATGAAAGT(TA)AT(TA)GT(TA)AT(TA)GG-3') complementary to the 5'-end of the NADH oxidase ( nox ) gene from Serpulina (Treponema) hyodysenteriae B204. The strains surveyed included 18 strains of S. hyodysenteriae , S. innocens strains B256 and 4/71, Treponema succinifaciens 6091, T. bryantii RUS-1, and 23 strains of uncharacterised (unclassified) intestinal spirochaetes. The uncharacterised spirochaetes had been isolated from the intestinal contents of different animal hosts (human, swine, avian, nutria). NADH oxidase was absent from T. succinifaciens and T. bryantii , but was present in every strain of S. hyodysenteriae , in the two recognised strains of S. innocens , and in every uncharacterised strain of intestinal spirochaete. Cells of four strains contained NADH oxidase activity but their DNA did not react with the probe. NADH oxidase is a common enzyme for the intestinal spirochaetes included in this survey. Keywords: NADH oxidase; spirochaete; intestinal bacteria; Serpulina hyodysenteriae ; nox ; intestinal microecology.

Fermenter cultivation of intestinal spirochaetes

We cultivated 6 strains of intestinal spirochaetes in a laboratory fermenter under constant conditions (medium, physical-chemical parameters) in a permanent gas flow. It has been possible to demonstrate that the application of the fermenter technique for the cultivation of spirochaetes is a suitable method which can be easily standardized.

Beta-Lactamase production by intestinal spirochaetes.

beta-Lactamase production was demonstrated in four of nineteen strains of intestinal spirochaetes isolated from human subjects. The enzyme was preferentially active against penicillins and was inhibited by clavulanic acid; it was membrane bound and non-inducible. No plasmids were detected in the intestinal spirochaetes and the beta-lactamase-production characteristic was not transferable to non-producing strains.

Spirochaete-mediated abnormal sex-ratio (SR) condition in Drosophila: a second virus associated with spirochaetes and its use in the study of the SR condition.

SUMMARYA class of abnormal sex-ratio (SR) condition in Drosophila characterized by the elimination of male zygotes is caused by infection with maternally transmitted SR-spirochaetes. A given spirochaete strain, WSR, was shown to have an associated DNA virus, spv-2, in a latent condition. This virus is induced to multiply when another DNA virus, previously designated spv-1, infects WSR spirochaetes; spv-1 is normally associated with another strain of SR-spirochaetes, NSR, and is in a manifest state. Spv-2 infects and lyses NSR, thus eliminating spirochaetes from fly hosts which, in these experiments, were D. melanogaster.The elimination of WSR through lysis by spv-1 results in the ‘curing’ of the SR-condition in the host flies. However, the elimination of NSR by spv-2 does not lead to the immediate elimination of the SR-condition in the host. A hypothesis is presented in favour of the view that a substance, androcidin, is produced by SR-spirochaetes and that this substance is actually responsible for the death of male zygotes. NSR may produce a more potent androcidin than WSR.

The in-vitro sensitivity of oral spirochaetes to metronidazole.

Ten pure strains of oral spirochaetes, having the same morphological and cultural characteristics as Treponema denticola, were isolated from 8 patients with acute necrotizing ulcerative gingivitis (ANUG). Penicillin and Metronidazole were tested for their ability to inhibit growth of these ten strains in vitro. The visual end point was 0.0–0.001 μ/ml for Penicillin and 0.1–0.01 μg/ml for Metronidazole. The significance of these results is discussed in relation to the aetiology and management of ANUG.

Tick-borne relapsing fever in Somaliland with special reference to the blood sedimentation rate.

Abstract 1. (1)|An account of the clinical features and complications of tick-borne relapsing fever in Somaliland has been given. 2. (2)|The Kahn test was negative in the eight cases examined. 3. (3)|In this series, neutrophilia was the exception rather than the rule. 4. (4)|B.S.R. estimations were made during, and for several weeks after, attacks. There was a tendency to a raised rate for several weeks after an attack, and to fluctuation in the B.S.R. without any corresponding clinical signs of activity. 5. (5)|It is believed that a latent infection persists in the body (possibly the brain) for a considerable period after all clinical signs have disappeared. An analogy is drawn to the latency and neurotropic character of Treponema pallidum . 6. (6)|Arsenical therapy was of benefit in some areas, while proving of no value whatsoever in other districts, the explanation probably being the occurrence of arsenic-resistant strains of spirochaetes in certain localities. 7. (7)|Penicillin did not prevent relapses.