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We report photometry and spectroscopy of the outburst of the young stellar object ESO-Halpha 99. The outburst was first noticed in Gaia alert Gaia18dvc and later by ATLAS. We have established the outburst light curve with archival ATLAS ``Orange'' filter photometry, Gaia data, new V-band photometry, and J, H, and K_s photometry from IRIS and UKIRT. The brightness has fluctuated several times near the light curve maximum. The TESS satellite observed ESO-Halpha 99 with high cadence during one of these minor minima and found brightness fluctuations on timescales of days and hours. Imaging with UKIRT shows the outline of an outflow cavity, and we find one knot of H_2 1-0 S(1) emission, now named MHO 1520, on the symmetry axis of this nebula, indicating recent collimated outflow activity from ESO-Halpha 99. Its pre-outburst SED shows a flat FIR spectrum, confirming its early evolutionary state and its similarity to other deeply embedded objects in the broader EXor class. The pre-outburst luminosity is 34 L_sun, a much higher luminosity than typical EXors, indicating that ESO-Halpha 99 may be a star of intermediate mass. Infrared and optical spectroscopy show a rich emission line spectrum, including HI lines, strong red CaII emission, as well as infrared CO bandhead emission, all characteristic EXors in the broadest sense. Comparison of the present spectra with an optical spectrum obtained in 1993, presumably in the quiescent state of the object, shows that during the present outburst the continuum component of the spectrum has increased notably more than the emission lines. The Halpha equivalent width during the outburst is down to one half of its 1993 level, and shock-excited emission lines are much less prominent., Accepted by The Astronomical Journal

A deep objective-prism survey for Hα emission stars towards the Canis Major star-forming clouds was performed. A total of 398 Hα emitters were detected, 353 of which are new detections. There is a strong concentration of these Hα emitters towards the molecular clouds surrounding the CMa OB1 association, and it is likely that these stars are young stellar objects recently born in the clouds. An additional population of Hα emitters is scattered all across the region, and probably includes unrelated foreground dMe stars and background Be stars. About 90% of the Hα emitters are detected by WISE, of which 75% was detected with usable photometry. When plotted in a WISE colour–colour diagram it appears that the majority are Class II YSOs. Coordinates and finding charts are provided for all the new stars, and coordinates for all the detections. We searched the Gaia-DR2 catalogue and from 334 Hα emission stars with useful parallaxes, we selected a subset of 98 stars that have parallax errors of less than 20% and nominal distances in the interval 1050 to 1350 pc that surrounds a strong peak at 1185 pc in the distance distribution. Similarly, Gaia distances were obtained for 51 OB-stars located towards Canis Major and selected with the same parallax errors as the Hα stars. We find a median distance for the OB stars of 1182 pc, in excellent correspondence with the distance from the Hα stars. Two known runaway stars are confirmed as members of the association. Finally, two new Herbig-Haro objects are identified.

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.

The aims of the current study were to collect intestinal spirochaetes (genus Brachyspira) from farmed and wild mallards (Anas platyrhynchos) and to identify and classify those isolates that phenotypically resembled Brachyspira hyodysenteriae, an enteric pathogen of pigs. The isolation rate of Brachyspira spp. was high from both farmed (93 %) and wild mallards (78 %). In wild mallards, it appeared that Brachyspira spp. were more likely to be found in migratory birds (multivariate analysis: RR = 1.8, 95 % CI 1.1-3.1) than in mallards sampled in a public park. Pure cultures of putative B. hyodysenteriae were obtained from 22 birds. All five isolates from farmed mallards and ten randomly selected isolates with this phenotype were used for further studies. All isolates from farmed mallards and two of the isolates from wild mallards were PCR-positive for the tlyA gene of B. hyodysenteriae. Two isolates from farmed mallards were selected for pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis. These isolates clustered with the type and reference strains of B. hyodysenteriae. 16S rDNA sequence analysis performed on 11 of the strains showed that they were all closely related to each other and to the B. hyodysenteriae-Brachyspira intermedia cluster. Three of the mallard isolates had 16S rDNA sequences that were identical to those of B. hyodysenteriae strains R1 and NIV-1 previously isolated from common rheas (Rhea americana). To conclude, the isolates from farmed mallards and two isolates from wild mallards were classified as B. hyodysenteriae based on the fact that they could not be differentiated by any of the applied methods from type, reference and field strains of B. hyodysenteriae. The remaining isolates could not be assigned irrefutably to any of the presently recognized Brachyspira species. These results point to a broader host spectrum of B. hyodysenteriae than is generally recognized, and to the presence in mallards of strongly beta-haemolytic and indole-producing spirochaetes that possess many, but not all, of the currently recognized characteristics of B. hyodysenteriae.

We present a deep survey of H? emission-line stars in the NGC 2264 region using wide-field objective prism films. We find 357 H? emission-line stars, of which 113 were previously detected, within an area of 3? ? 3? centered on the Cone Nebula, with a majority of stars being concentrated in a dense cluster at the center of the region. We present a large-scale CO map of NGC 2264 and find a strong correlation between the cluster of H? emission-line stars and the most massive core in the cloud complex. A more extended halo of stars poorly correlated with the gas may represent stars that have drifted away from their birth place, suggesting that star formation has taken place in NGC 2264 for several million years.

The composition of the initial bacterial flora of pork and the development of the flora after storage at +4 degrees C for 4 days were analysed by amplification, cloning and sequencing of 16S rDNA. A total of 122 clones were obtained, with lengths ofor =400 nucleotides andor =95% similarity to database sequences. Nineteen clones were similar to sequences in database not assigned to any genera. Fourteen different genera were represented in clones from fresh meat, with 36.5% of the clones most resembling Acinetobacter and 17.3% resembling Staphylococcus and Macrococcus. After storage, the clones were composed of six different genera, with 44.3% resembling Pseudomonas, 17.1% resembling Aeromonas and only 14.3% resembling Acinetobacter. This study shows that the overall pattern of the initial and chill-stored pork flora, as shown by a molecular approach, was in agreement with results obtained in previous studies using traditional cultivation methods.

We present a deep survey of Halpha emission-line stars in the M42 region using wide-field objective prism films. A total of 1699 Halpha emission-line stars were identified, of which 1025 were previously unknown, within an area of 5.5 x 5.5 degrees centred on the Trapezium Cluster. We present Halpha strength estimates, positions, and JHKs photometry extracted from 2MASS, and comparisons to previous surveys. The spatial distribution of the bulk of the stars follows the molecular cloud as seen in CO and these stars are likely to belong to the very young population of stars associated with the Orion Nebula Cluster. Additionally, there is a scattered population of Halpha emission-line stars distributed all over the region surveyed, which may consist partly of foreground stars associated with the young NGC 1980 cluster, as well as some foreground and background dMe or Be stars. The present catalogue adds a large number of candidate low-mass young stars belonging to the Orion population, selected independently of their infrared excess or X-ray emission., Comment: 38 pages, 18 figures. Published in Astronomy & Astrophysics v3: chart for ESO-HA 1639 corrected. Typos in Table 2 corrected. Photometry for ESO-HA 1639 in Table 2 corrected

Brachyspira spp.were isolated from 21 of 32 sampled dogs (66%) in a colony of Swedish beagle dogs with a history of diarrhea and from 3 of 17 Swedish pet dogs (17%) with diarrhea. All Swedish isolates were weakly β-hemolytic and gave a negative indole reaction. Eighty-eight percent showed negative α-galactosidase and hippurate reactions, but a positive β-glucosidase reaction. Two isolates were hippurate positive with a negative β-glucosidase reaction. One additional German isolate diverged by showing a positive indole reaction in combination with a positive hippurate reaction. Sequencing of 16S rDNA indicated that the hippurate-positive isolates belonged to the speciesBrachyspira pilosicoli. Four representative isolates were examined using pulsed-field gel electrophoresis (PFGE) and compared with six reference strains and five porcine isolates ofBrachyspiraspp. The canine isolates clustered together in the PFGE analysis. Necropsy examination of a culture-positiveB. pilosicolicolony-raised beagle dog revealed macro- and microscopical lesions of colitis with numerous spiral-shaped bacteria in the lumens of the crypts, in goblet cells and within the colonic epithelium.

Aims: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. Methods and Results:Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8–357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. Conclusions: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. Significance and Impact of the Study: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.

Sixty-four strains of Lactobacillus were isolated from fermentation samples from 23 malt whisky distilleries located in the major whisky producing regions of Scotland. The strains were assigned to 26 ribotype patterns. Strains of some ribotype patterns were widely distributed and recovered from distilleries throughout Scotland, while strains representing other ribotypes were particular to a specific region or even a certain distillery. Repeated sampling of a single distillery over a 12 month period showed that the range of bacteria present, as indicated by ribotyping, was stable, but was influenced by changes in malt supply and the period of closure for annual maintenance. Partial 16S rDNA sequence analysis of ribotype representatives revealed Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus pentosus as the major species present in the distilleries; however, four isolates could not be identified by this procedure. Determination of the full 16S rDNA gene sequence from one of these isolates (strain R7-84) revealed >98·5% similarity to Lactobacillus buchneri and its phylogenetic neighbours. DNA from two other strains showed greater than 70% hybridization to DNA from R7-84 under non-stringent renaturation conditions and DNA from strain R7-84 shared less than 65% hybridization with members of the L. buchneri group. It is proposed that these three strains should be placed in a new species for which the name Lactobacillus ferintoshensis represented by the type strain R7-84T is suggested.

The Mycoplasma lipophilum cluster (Weisburg et al. 1989) in the hominis group of the mollicutes is re-evaluated in this work to update the phylogenetic framework for classification of species within the genus Mycoplasma. Therefore, sequences of the 16S rRNA gene were determined from previously described species, and 11 were found to be closely related to the M. lipophilum cluster. A selection of members of the other hitherto defined clusters of the hominis group was included for phylogenetic analysis, revealing that the classical M. lipophilum cluster could be re-organized into two clusters, namely the M. lipophilum cluster and the Mycoplasma bovis cluster. The former was found to contain two species, while the latter contained 20 species. The two clusters were closely related, sharing an ancestral branch with the Mycoplasma synoviae cluster. Furthermore, the M. bovis cluster could be divided into subclusters. Interestingly, two species, Mycoplasma equigenitalium and Mycoplasma elephantis, formed a distinct and early branch of the M. lipophilum, M. bovis and M. synoviae clusters. This entity was termed the M. equigenitalium cluster. The clusters and subclusters could be verified by using neighbour-joining and maximum-likelihood analyses on a variety of data sets, bootstrap calculations, secondary structure analysis and signature nucleotides. Therefore, the new 16S rDNA data presented in this work were used to re-evaluate the M. lipophilum cluster, leading to the definition of two additional clusters. At present, the mollicutes belonging to the hominis group can be classified into ten evolutionary lineages.

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly beta-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.

Thirty-three strains previously classified into 11 species in the bacterial family Moraxellaceae were subjected to phylogenetic analysis based on 16S rRNA sequences. The family Moraxellaceae formed a distinct clade consisting of four phylogenetic groups as judged from branch lengths, bootstrap values and signature nucleotides. Group I contained the classical moraxellae and strains of the coccal moraxellae, previously known as Branhamella, with 16S rRNA similarity ofor = 95%. A further division of group I into five tentative clusters is discussed. Group II consisted of two strains representing Moraxella atlantae and Moraxella osloensis. These strains were only distantly related to each other (93.4%) and also to the other members of the Moraxellaceae (or = 93%). Therefore, reasons for reclassification of these species into separate and new genera are discussed. Group III harboured strains of the genus Psychrobacter and strain 752/52 of [Moraxella] phenylpyruvica. This strain of [M.] phenylpyruvica formed an early branch from the group III line of descent. Interestingly, a distant relationship was found between Psychrobacter phenylpyruvicus strain ATCC 23333T (formerly classified as [M.] phenylpyruvica) and [M.] phenylpyruvica strain 752/52, exhibiting less than 96% nucleotide similarity between their 16S rRNA sequences. The establishment of a new genus for [M.] phenylpyruvica strain 752/52 is therefore suggested. Group IV contained only two strains of the genus Acinetobacter. Strategies for the development of diagnostic probes and distinctive sequences for 16S rRNA-based species-specific assays within group I are suggested. Although these findings add to the classificatory placements within the Moraxellaceae, analysis of a more comprehensive selection of strains is still needed to obtain a complete classification system within this family.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.

An approach for real-time DNA sequencing without the need for electrophoresis has been developed. The approach relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate (PPi) detection assay (ELIDA) (Nyrén, P. (1987) Anal. Biochem. 167, 235-238). The PPi formed in the DNA polymerase reaction is converted to ATP by ATP sulfurylase and the ATP production is continuously monitored by the firefly luciferase. In the sequencing procedure, immobilized single-stranded template was used in a repeated cycle of deoxynucleotide extension. Real-time signals in the ELIDA, proportional to the amount of incorporated nucleotide, were observed when complementary bases were incorporated. An increased signal-to-noise ratio was obtained by substitution of deoxyadenosine alpha-thiotriphosphate (dATP alpha S) for the natural deoxyadenosine triphosphate, dATP alpha S is efficiently used by the DNA polymerase, but is not recognized by the luciferase. As a model, 15 bases of a single-stranded PCR product were sequenced. The possibility for parallel processing of many samples in an automated manner is discussed.

Almost complete (>96%) 16S rRNA sequences from nine ruminant mycoplasmas have been determined by solid-phase DNA sequencing. Polymorphisms were found in four of the 16S rRNA sequences, which indicated the existence of two different rRNA operons. Seven polymorphisms were found in Mycoplasma agalactiae, three were found in Mycoplasma bovis, one was found in Mycoplasma alkalescens, and one was found in Mycoplasma bovirhinis. The sequence data were used for construction of phylogenetic trees. All but one of the ruminant mycoplasmas sequenced in this work clustered in the hominis group. A close relationship was found between M. agalactiae and M. bovis, with a 99% nucleotide similarity between their 16S rRNA sequences. They were also found to be members of the Mycoplasma lipophilum cluster of the hominis group. Furthermore, the 16S rRNA comparisons showed that Mycoplasma alkalescens and Mycoplasma canadense are closely related (>98.5%), and these species were found to cluster in the Mycoplasma hominis cluster of the hominis group. Interestingly, M. bovirhinis grouped in a new phylogenetic cluster of the hominis group. The new cluster, which was supported by bootstrap percentage values, signature nucleotide analysis, and higher-order structural elements, was named the Mycoplasma synoviae cluster. Mycoplasma bovoculi, Mycoplasma conjunctivae, and Mycoplasma ovipneumoniae clustered in the Mycoplasma neurolyticum cluster of the hominis group. Mycoplasma alvi clustered with Mycoplasma pirum in the M. pneumoniae cluster of the pneumoniae group.

The so-called Mycoplasma mycoides cluster consists of six species or subspecies of mycoplasmas (Mollicutes). These species are pathogenic for ruminants and some of them are of great concern in veterinary medicine. The members of the M. mycoides cluster have two rRNA operons (rrnA and rrnB). The nucleotide sequences of the 16S rRNA genes of 10 strains, representing all of the known species and subspecies of the M. mycoides cluster, were determined by direct automated solid-phase DNA sequencing. The sequences of both rRNA operons were determined by a novel strategy involving in vitro amplification by PCR with one operon-specific primer pair and one general primer pair. Interestingly, sequence differences (polymorphisms) between the two operons were observed for all strains. Two strains of M. capricolum subsp. capripneumoniae were sequenced, and 15 polymorphisms were found in the type strain (F38) and 17 polymorphisms were found in the other strain (4/2LC). Eight polymorphisms were found in the 16S rRNA genes of the M. mycoides subsp. mycoides small-colony type, and sequence length variations in a poly(A) region were observed in the 16S rRNA genes of the two operons of this species. Secondary-structure analysis showed that polymorphisms were present in both stem and loop regions. The nucleotide substitutions in the polymorphic sites of the stem regions often resulted in a change from a canonical to a noncanonical base pairing or vice versa. A compensatory mutation was never observed in the other nucleotide of the base pair. Phylogenetic analysis based on the 16S rRNA sequences indicated that Mycoplasma sp. strain PG50 should be included in the M. capricolum species group. Furthermore, the 16S rRNA sequences of M. mycoides subsp. capri and the M. mycoides subsp. mycoides large-colony type were 99.9% identical. We therefore suggest that these species be reclassified in a common species group (for instance, "Mycoplasma capri") distinct from the M. mycoides subsp. mycoides small-colony type, which formed an intermediate branch between the M. capricolum species group and the M. capri species group.

Four type or reference strains and twenty-two field strains of intestinal spirochetes isolated from Swedish pig herds were subjected to phylogenetic analysis based on 16S rRNA sequences. Almost complete (>95%) 16S rRNA sequences were obtained by solid-phase DNA sequencing of in vitro-amplified rRNA genes. The genotypic patterns were compared with a previously proposed biochemical classification scheme, comprising beta-hemolysis, indole production, hippurate hydrolysis, and alpha-galactosidase, alpha-glucosidase, and beta-glucosidase activities. Comparison of the small-subunit rRNA sequences showed that the strains of the genus Serpulina were closely related. Phylogenetic trees were constructed, and three clusters were observed. This was also confirmed by signature nucleotide analysis of the serpulinas. The indole-producing strains, including the strains of S. hyodysenteriae and some weakly beta-hemolytic Serpulina strains, formed one cluster. A second cluster comprised weakly beta-hemolytic strains that showed beta-galactosidase activity but lacked indole production and hippurate-hydrolyzing capacity. The second cluster contained two subclusters with similar phenotypic profiles. A third cluster involved strains that possessed a hippurate-hydrolyzing capacity which was distinct from that of the former two clusters, because of 17 unique nucleotide positions of the 16S rRNA gene. Interestingly, the strains of this third cluster were found likely to have a 16S rRNA structure in the V2 region of the molecule different from that of the serpulinas belonging to the other clusters. As a consequence of these findings, we propose that the intestinal spirochetes of this phenotype (i.e., P43/6/78-like strains) should be regarded as a separate Serpulina species. Furthermore, this cluster was found to be by far the most homogeneous one. In conclusion, the biochemical classification of porcine intestinal spirochetes was comparable to that by phylogenetic analysis based on 16S rRNA sequences..

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum-like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using HindIII and EcoRI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917 T , Lact. pentosus ATCC 8041 T , Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei) had identical 16S ribosomal DNA sequences (Lact. plantarum/Lact. pentosus). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensus stricto ; three of these strains had more than 60% DNA : DNA homology to the type strain of Lact. plantarum, and one had less than 50% DNA : DNA homology to Lact. plantarum ATCC 14917 T . Two strains had fragment patterns similar to the type strain of Lact. pentosus, and they had more than 80% DNA : DNA homology to Lact. pentosus ATCC 8041 T . One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of the phenotype and source of isolation. RFLP of the 16S rRNA genes using EcoRI and HindIII might be used for species recognition of Lact. plantarum, but seems less suitable for strain typing.

Twenty-one putative Serpulina strains, representing six proposed biochemical groups, were selected for phylogenetic studies based on 16S rRNA sequencing. The biochemical groups were distinguished by the degree of β-haemolysis, indole production, hippurate hydrolysis and a-galactosidase-, and β-glucosidase activity. The 16S rRNA sequences of the U2 to U5 region, including three evolutionarily variable regions, from representatives of each biochemical group were determined by automated solid phase DNA sequencing after in vitro amplification by the polymerase chain reaction ( PCR ). The sequences generated were 532 nucleotides in length. Sequence alignments showed that all the strains were closely related, with six informative positions in the region sequenced. A dendrogram was constructed from these data and compared with the tentative biochemical classification. The results support the proposed biochemical classification and indicate that at least five genetic variants of the genus Serpulina can be identified.

Seven Swedish isolates of Ehrlichia species from the blood of four dogs and three horses with clinical granulocytic ehrlichiosis, were identified by direct solid phase DNA sequencing of polymerase chain reaction (PCR) products from the 16S rRNA gene. The amplified DNA fragments were produced with primers complementary to the universal regions, Ul, U2, U5 and U8 of the 16S rRNA molecule. Identical sequences were obtained from all seven isolates. This nucleotide sequence was similar to the sequences deposited in GenBank for Ehrlichia phagocytophila and E equi . The sequence of the Swedish ehrlichiae differed in two nucleotide positions from the E phagocytophila sequence and in three positions from the E equi sequence, and it is tentatively proposed that it is a subspecies of one of these two. The alignment of the sequence of the Swedish isolates with a recently deposited sequence from human cases of ehrlichiosis in the USA revealed 100 per cent identity in a segment of about 1400 bp.

Summary Restriction Fragment Length Polymorphisms (RFLPs) of the 16S rRNA gene were determined for fourteen field strains originally labelled Lactobacillus reuteri , four Lactobacillus fermentum strains, and eight type and reference strains of Lactobacillus . The strains were mainly originating from intestinal mucosa. The probe was a 420 bp internal fragment of a Lactobacillus reuteri 16S rRNA gene, amplified by Polymerase Chain Reaction (PCR). The patterns were obtained by cleavage of the chromosomal DNAs by the restriction endonucleases E co RI and Hind III separately. Unweighted Pair Group Method, using arithmetic Averages (UPGMA) based on Jaccard coefficients (S J ) of the ribopatterns resolved five clusters at the 30% similarity level. The L. reuteri field strains formed two clusters according to their source of isolation; strains originating from rat formed one cluster while those from pig and human strains formed the other cluster. These strains could not be affiliated to the type strain of L. reuteri . However, two field strains, classified by phenotypically means as L. fermentum , clustered together with the type strain of this species. The numbers of 16S rRNA genes of the L. reuteri strains were, depending on the strain, determined to be at least five through seven. The ribopatterns were compared to the results of DNA:DNA hybridization and partial sequencing of the 16S rDNA genes. All but one of the L. reuteri field strains had more than 70% DNA similarity and identical 16S rDNA sequence to the type strain L. reuteri DSM 20016 T . Two of the L. fermentum field strains had identical 16S rDNA sequence and more than 70% DNA similarity to L. fermentum ATCC 14931 T . Ribotyping appears not to be a means to identify L. reuteri at the species level but eventually this is possible with L. fermentum .

To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes.

A solid phase DNA sequencing method for non-radioactive detection of single base changes without the need for electrophoresis is presented. The concept relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (P. Nyren, 1987, Anal. Biochem. 167, 235-238). Immobilized DNA fragments amplified with the polymerase chain reaction are used as template. A detection primer is annealed in front of the mutation and four aliquots of this mixture are incubated with DNA polymerase and one of the four different dideoxynucleotides. The presence or absence of an incorporated dideoxynucleotide is thereafter monitored by the release of inorganic pyrophosphate during the following primer extension step. We show that the concept can be used for sequencing of single bases as well as step-wise analysis of several subsequent bases.

The influence of the cleanliness of cows' stalls on the cows' own cleanliness and on the health of their hooves was investigated in 52 dairy cows tied in stalls with long stanchions from 2 October 1989 to 7 May 1990 in one Swedish dairy herd. Twenty-six of the cows were provided with electric cow trainers throughout this period, 14 did not have trainers and 12 had trainers only for the period between 7 February and 7 May. The hooves of the hind feet were evaluated for Dermatitis interdigitalis (interdigital dermatitis) and Erosio ungulae (heel-horn erosion) on 2 October, 7 February and 7 May, and on this last occasion the dry matter content of the sole horn was measured. The average number of cow-pats deposited on the floors of the stalls with electric trainers was less than one-third of the number in the stalls without them, and the cows in stalls with trainers were approximately 30% cleaner. Between October and February, heel-horn erosion increased significantly only in the cows without electric trainers, and between February and May, heel-horn erosion decreased only in the cows which were provided with trainers on 7 February. The prevalence of inter-digital dermatitis decreased in all the cows throughout the period of housing and there was no significant difference in prevalence between the different groups. The dry matter content of the sole horn was significantly greater in the cows which had electric trainers than in the cows without trainers, and the difference was significant even in the cows which had the trainers only from February to May. There was a significant negative correlation between the dry matter content of the sole horn and the level of heel-horn erosion.

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rabnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rabnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.

It has been suggested that canine intestinal spirochaetes consist of Brachyspira pilosicoli and a group of strains that has been provisionally designated 'Brachyspira canis'. The purpose of the present study was to compare 22 spirochaete isolates that were obtained from intestinal specimens of dogs in Sweden (n = 12), Norway (n = 4), the United States (n = 3), Australia (n = 2) and Germany (n = 1) with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences. In an evolutionary tree derived from 16S rDNA sequences, the canine isolates grouped into three clusters. One cluster included the type strain of porcine B. pilosicoli, whereas a second larger cluster, which was monophyletic, contained a canine strain that was identified previously as 'B. canis'. The third cluster consisted of three canine isolates of Scandinavian origin, which grouped together with the type strain of the species Brachyspira alvinipulli (pathogenic to chicken). These three genotypes, which were identified on the basis of 16S rDNA sequences, corresponded to four phenotypic groups based on biochemical testing. Two biochemical tests, hippurate hydrolysis and alpha-galactosidase production, were sufficient for rapid identification of each canine cluster.

Mycoplasma mycoides subsp. mycoidesSC (MmymySC)is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease in cattle. The genome of Mmymy SC type strain PG1T has been sequenced to map all the genes and to facilitate further studies regarding the cell function of the organism and CBPP. The genome is characterized by a single circular chromosome of 1,211,703 bp with the lowest G+C content (24 mole%)and the highest density of insertion sequences (13% of the genome size)of all sequenced bacterial genomes. The genome contains 985 putative genes, of which 72 are part of insertion sequences and encode transposases. Anomalies in the GC-skew pattern and the presence of large repetitive sequences indicate a high genomic plasticity. A variety of potential virulence factors was identified, including genes encoding putative variable surface proteins and enzymes and transport proteins responsible for the production of hydrogen peroxide and the capsule, which is believed to have toxic effects on the animal.

A new insertion sequence, ISMmy1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC). The occurrence of ISMmy1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG1(T), the vaccine strain T1Sr49, and the strain Afadé, which all had unique patterns. ISMmy1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta(T) while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy1. This observation suggests horizontal transfer between MmymySC and M. bovis.

Four strains of aerobic, endospore-forming bacteria were isolated from the inner tissues of healthy cotton plants (Gossypium sp., Dushanbe, Tajikistan). The organisms had identical randomly amplified polymorphic DNA patterns that distinguished them from other bacilli that are commonly isolated from plant tissues, e.g. Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis. PCR amplification of 16S-23S rRNA spacer regions suggested that the four strains could be assigned to two highly related taxa, which correlated with differences in cell morphology. However, the cloned spacer region provided a simple and specific hybridization probe for all four strains. The virtually complete 16S rDNA sequences were prepared for representatives of the two groups (strains 2DT(T) and 12DX) and differed by only two bases, thus supporting classification of the four strains in a single taxon at the species level. Phylogenetic analyses indicated that strain 2DT(T) belonged to the genus Bacillus and was most closely related to Bacillus sporothermodurans DSM 10599T with a sequence similarity of 94.8%. It is concluded that the four strains belong to a novel species of Bacillus for which the name Bacillus endophyticus sp. nov. is proposed. The type strain is 2DT(T) (= UCM B-5715T = CIP 106778T).

Many commonly used bacteriological terms are far from being well defined and in scientific papers these terms may be used with different meanings15, 89. One of the purposes of bacterial taxonomy is to avoid confusion by creating a database with a common language for bacteriologists. The word taxonomy is derived from the Greek words taxis and nomos, which mean arrangement or order and law, respectively. Microbial taxonomy includes three different, but related areas, namely classification, nomenclature and identification2. Taxonomy can be regarded as a complete system for organisation of information about organisms and classification is defined as the theories and principles for arranging organisms into hierarchic groups of the above database. Taxon (pl. taxa) is defined as a group of organisms of any rank in the taxonomic hierarchy62. For instance, species, family and domain are taxa and these taxa are ranked according to their level of inclusiveness. Nomenclature refers to the process of naming taxa on the basis of similarities or relations, which for bacteria is regulated by the so-called Bacteriological Code72. Identification is the practical application of a classification scheme to establish the identity of an isolate. Systematics is sometimes used as a synonym for taxonomy, but phylogenetic aspects are often also included in systematics74.

DNA was extracted from colonic biopsies of 33 patients with and three without evidence of intestinal spirochetosis (IS) in the large bowel. The biopsies were subjected to PCR. A pair of primers, generating a 207 bp fragment, were designed to detect specifically the 16S rDNA gene ofBrachyspira. PCR products of the expected size were obtained from 33 samples with histologic evidence of IS. The PCR amplicons were used for sequencing. The sequences obtained were aligned to the corresponding 16S rRNA sequences of five type strains ofBrachyspira. The sequences of 23 PCR products were 99–100% identical with the correspond-ingB.aalborgitype strain sequence. Two cases showed 99–100% sequence similarity with the type strain ofB.pilosicoliP43/6/78. Six cases could not be referred to any of the known species ofBrachyspira. Two PCR products gave incomplete sequences.

The bacterial flora of the intestine and the bacteria found in liver, mesenteric lymph nodes, portal and arterial blood after D-galactosamine-induced liver injury, with and without pretreatment with Lactobacillus plantarum DSM 9843, were studied in the rat. Dominating representatives were identified to species level by 16S rDNA sequencing and typed by randomly amplified polymorphic DNA (RAPD) and by restriction endonuclease analysis (REA) for strain definition. It was proven that bacterial strains from the intestine occur at extraintestinal sites after liver injury. Lactobacillus spp. dominated the intestinal flora and were also the most frequently found genus in the liver and the mesenteric lymph nodes. Some of the blood isolates, identified as Staphylococcus aureus, Proteus vulgaris and Bacteroides merdae, were not found as a dominating part of the mucosal flora. Treatment with L. plantarum before liver injury decreased translocation and made the intestinal flora increasingly dominated by lactobacilli.

The nucleotide sequences of the 16S rRNA genes from the type strains of three seal mycoplasmas, Mycoplasma phocicerebrale, Mycoplasma phocae and Mycoplasma phocirhinis (formerly Mycoplasma phocacerebrale, Mycoplasma phocidae and Mycoplasma phocarhinis, respectively), were determined by direct DNA cycle sequencing. Polymorphisms were found in all three 16S rRNA gene sequences, showing the existence of two different rRNA operons. In M. phocae, a length difference was found between the operons, caused by an insertion or a deletion of an adenosine in one of the operons. The sequence information was used to construct phylogenetic trees. All three species were found to belong to the hominis group, but to different clusters. M. phocicerebrale and M. phocae were found to be members of the Mycoplasma hominis cluster, within which M. phocicerebrale grouped in the Mycoplasma alkalescens subcluster. M. phocirhinis was found to be a member of the Mycoplasma bovigenitalium subcluster of the Mycoplasma bovis cluster. The 16S rRNA gene sequences of all hitherto validly described species within the M. hominis and M. bovis clusters have now been determined.

Sixty-four strains of Lactobacillus were isolated from fermentation samples from 23 malt whisky distilleries located in the major whisky producing regions of Scotland. The strains were assigned to 26 ribotype patterns. Strains of some ribotype patterns were widely distributed and recovered from distilleries throughout Scotland, while strains representing other ribotypes were particular to a specific region or even a certain distillery. Repeated sampling of a single distillery over a 12 month period showed that the range of bacteria present, as indicated by ribotyping, was stable, but was influenced by changes in malt supply and the period of closure for annual maintenance. Partial 16S rDNA sequence analysis of ribotype representatives revealed Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus pentosus as the major species present in the distilleries; however, four isolates could not be identified by this procedure. Determination of the full 16S rDNA gene sequence from one of these isolates (strain R7-84) revealed98.5% similarity to Lactobacillus buchneri and its phylogenetic neighbours. DNA from two other strains showed greater than 70% hybridization to DNA from R7-84 under non-stringent renaturation conditions and DNA from strain R7-84 shared less than 65% hybridization with members of the L. buchneri group. It is proposed that these three strains should be placed in a new species for which the name Lactobacillus ferintoshensis represented by the type strain R7-84(T) is suggested.

A novel bacterial strain (171544T) was recently isolated from silage and was classified in the genus Bacillus by 16S rDNA sequence analysis. Additional silage samples have been investigated in the present study and four organisms resembling strain 171544T were isolated. Phenotypic and genotypic characterization of these bacteria showed that they constitute a new species of the genus Bacillus. This taxon was positioned in the family Bacillaceae on the basis of evolutionary distance trees using 16S rDNA sequences. Bacillus circulans, Bacillus firmus and Bacillus benzoevorans were the most closely related species with 165 rDNA similarities of 97.2, 96.3 and 95.9%, respectively. All five silage isolates shared a higher order structural feature in the 3' region of the 16S rRNA gene comprising an extension to helix 49 of 24 bp and highly similar random amplified polymorphic DNA patterns that distinguished them from the type strains of B. circulans and B. firmus. Moreover, they possessed a unique pattern of phenotypic features including subterminally or terminally located endospores which distinctly swelled the sporangium, strictly aerobic metabolism but with the ability to utilize nitrate as a terminal electron acceptor under anaerobic conditions, and hydrolysis of casein but not starch. The name Bacillus siralis is therefore proposed for this new taxon. The type strain of B. siralis is strain 171544T (= NCIMB 13601T = CIP 106295T).

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.

Intestinal spirochetes (Brachyspira spp.) are causative agents of intestinal disorders in animals and humans. Phylogenetic analysis of cloned 16S rRNA genes from biopsies of the intestinal mucosa of the colon from two Swedish 60-years old adults without clinical symptoms revealed the presence of intestinal spirochetes. Seventeen clones from two individuals and 11 reference strains were analyzed and the intestinal spirochetes could be divided into two lineages, the Brachyspira aalborgi and the Brachyspira hyodysenteriae lineages. All of the clones grouped in the B. aalborgi lineage. Moreover, the B. aalborgi lineage could be divided into three distinct phylogenetic clusters as confirmed by bootstrap and signature nucleotide analysis. The first cluster comprised 6 clones and the type strain B. aalborgi NCTC 11492T. The cluster 1 showed a 16S rRNA gene similarity of 99.4-99.9%. This cluster also harbored the only other strain of B. aalborgi isolated so far, namely strain W1, which was subjected to phylogenetic analysis in this work. The second cluster harbored 9 clones with a 98.7 to 99.5% range of 16S rDNA similarity to the B. aalborgi cluster 1. Two clones branched distinct and early of the B. aalborgi line forming the third cluster and was found to be 98.7% similar to cluster 1 and 98.3-99.1% to cluster 2. Interestingly, this shows that considerable variation of intestinal spirochetes can be found as constituents of the colonic microbiota in humans, genetically resembling B. aalborgi. The presented data aid significantly to the diagnostic and taxonomic work on these organisms.

In this study we report on the isolation and characterization of the intestinal spirochete Brachyspira aalborgi using human mucosal biopsy specimens taken from the colon of a young adult male with intestinal spirochetosis. A selective medium, containing 400 μg of spectinomycin/ml and 5 μg of polymyxin/ml was used for the isolation procedure. A high degree of similarity, in terms of phenotypic properties and 16S ribosomal DNA sequence, was observed between the isolated strain, named W1, and the type strain, 513A, of B. aalborgi . A similarity of 99.7% in the nucleotide sequence was found between W1 and 513A T , based on the almost-complete gene. A short segment of the 16S rRNA gene was amplified by PCR using genetic material enriched from paraffin-embedded biopsy specimens, which were taken from the patient on two occasions. The products showed 16S rRNA gene sequences virtually identical to that of strain 513A T in the actual region. Immunohistochemistry was performed on the colonic biopsy specimens with a polyclonal antibody raised against an intestinal spirochete isolated in a previous case of human intestinal spirochetosis. The antibody reacted strongly with the spirochete on the luminal epithelium. No immune reaction was seen within or below the surface epithelium. Routine histology did not reveal signs of colitis. Electron microscopy showed spirochetes attached end-on to the colonic mucosal surface. The isolate grew poorly on a commonly used selective medium for intestinal spirochetes, which may explain previous failures to isolate B. aalborgi .

The fastidious nature of the mollicutes (mycoplasmas), their lack of a classic bacterial cell wall, and their very small genome, make phylogenetic placements of new species in this enlarging group of prokaryotes an important and valuable aid in their classification. In this report we have determined the phylogeny of the Mycoplasma hominis cluster of the hominis group. The 16S rDNA sequences from several previously described Mycoplasma species were determined and ten species were found to belong to the M. hominis cluster. With almost complete sequences available, the phylogenetic analysis revealed that the M. hominis cluster currently comprises 19 species, forming a distinct clade as judged from branch lengths, bootstrap percentage values, nucleotide signature analysis, and structural elements in the 16S rRNA molecule. The 16S rRNA gene sequences of species in the M. hominis cluster were found to be > or = 94% similar and the range within which similarities can be used in the classification of new species is discussed. Members of the M. hominis cluster all share a major biochemical property of M. hominis, in that they hydrolyse arginine and are incapable of fermenting glucose. This consistency in phenotypic pattern has not been found in any of the other phylogenetic clusters of the hominis group. Two species, the non-cultivable agent of Grey Lung disease in rodents (tentatively named 'Candidatus Mycoplasma ravipulmonis') and the avian species Mycoplasma gypis strain B1/T1T, were regarded as close relatives to the M. hominis cluster, but are clearly separated from the species of this cluster. Both species formed early branches of the M. hominis cluster and should be regarded as individual lines containing one species.

The sequence of the RNase P RNA gene (rnpB) was determined for 60 strains representing all nine species in the family Chlamydiaceae and for the related Chlamydiales species, Parachlamydia acanthamoebae and Simkania negevensis. These sequences were used to infer evolutionary relationships among the Chlamydiaceae. The analysis separated Chlamydophila and Chlamydia into two lineages, with Chlamydophila forming three distinct clusters: the Chlamydophila pneumoniae strains; the Chlamydophila pecorum strains; and a third cluster comprising the species Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae and Chlamydophila felis. The Chlamydia line of descent contained two clusters, with the Chlamydia suis strains distinctly separated from strains of Chlamydia trachomatis and Chlamydia muridarum. This analysis indicated that the rnpB sequence and structure are distinctive markers for species in the Chlamydiaceae. It was also demonstrated that the RNase P RNA derived from Chlamydia trachomatis is able to cleave a tRNA precursor in the absence of protein. These findings are discussed in relation to the structure of Chlamydia RNase P RNA.

As contagious bovine pleuropneumonia (CBPP) is spreading fast in many African countries, there is an increasing demand for rapid and sensitive diagnostic methods that can be used to confirm the initial diagnosis based on clinical symptoms or pathological findings. Two PCR-based diagnostic systems for identification of the infectious agent, Mycoplasma mycoides subsp. mycoides SC ( M. mycoides SC), in various samples are presented. Both systems involve group-specific amplification of the two 16S rRNA genes from mycoplasmas of the M. mycoides cluster. The laser-induced fluorescence assay is based on a unique sequence length difference between the two 16S rRNA genes in M. mycoides SC. This region was amplified by PCR, and the products were separated by polyacrylamide gel electrophoresis in a DNA sequencer. The resulting electropherogram showed two peaks for strains of M. mycoides SC and one peak for all other members of the M. mycoides cluster. The second system was based on restriction endonuclease analysis and agarose gel electrophoresis. Restriction of amplicons from a region containing a polymorphism, which is found in M. mycoides SC only, resulted in an extra band on the agarose gel because an Alu I site is lacking in the rrnA operon. Specimens from cows with postmortem signs of CBPP were analyzed with the two PCR systems. M. mycoides SC was clearly identified in pleural fluid and lung tissue, and the methods were found to be robust and rapid. The results were in agreement with those obtained by conventional diagnostic techniques.

The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined. Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes). This cluster will be referred to as the M. fastidiosum cluster. Interestingly, the M. fastidiosum cluster formed a sister lineage to the haemotrophic bacteria, Eperythrozoon spp. and Haemobartonella spp. The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures. Thus, the members of the M. fastidiosum cluster are the closest known relatives to the haemotrophic bacteria. Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.

Almost complete 16S rRNA gene sequences were generated for the type strains of the obligate insect pathogens Bacillus lentimorbus and Bacillus popilliae and a second strain of Bacillus popilliae (NRRL B-4081) received as ‘ Bacillus popilliae var. melolonthae’. A phylogenetic tree was constructed which grouped these strains into a well defined subcluster within the genus Paenibacillus. Bacillus popilliae NRRL B-4081 occupied an intermediate position between the type strains of Bacillus lentimorbus and Bacillus popilliae but with a marked clustering to the latter. The phylogenetic assignment of these strains to Paenibacillus is in contrast to earlier studies which placed these bacteria in the genus Bacillus, close to Bacillus subtilis. Indeed, the rRNA sequences generated in this study share less than 88% similarity to the deposited sequences for Bacillus popilliae ATCC 14706Tand Bacillus lentimorbus ATCC 14707T. The results obtained by using different tree algorithms, bootstrap analysis, branch lengths and verification by signature nucleotide analysis supported the reclassification of these species in the genus Paenibacillus as Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov.