Evolutionary relationships among Chlamydophila abortus variant strains inferred by rRNA secondary structure-based phylogeny.

The evolutionary relationships among known Chlamydophila abortus variant strains including the LLG and POS, previously identified as being highly distinct, were investigated based on rRNA secondary structure information. PCR-amplified overlapping fragments of the 16S, 16S-23S intergenic spacer (IS), and 23S domain I rRNAs were subjected to cloning and sequencing. Secondary structure analysis revealed the presence of transitional single nucleotide variations (SNVs), two of which occurred in loops, while seven in stem regions that did not result in compensatory substitutions. Notably, only two SNVs, in 16S and 23S, occurred within evolutionary variable regions. Maximum likelihood and Bayesian phylogeny reconstructions revealed that C. abortus strains could be regarded as representing two distinct lineages, one including the "classical" C. abortus strains and the other the "LLG/POS variant", with the type strain B577(T) possibly representing an intermediate of the two lineages. The two C. abortus lineages shared three unique (apomorphic) characters in the 23S domain I and 16S-23S IS, but interestingly lacked synapomorphies in the 16S rRNA. The two lineages could be distinguished on the basis of eight positions; four of these comprised residues that appeared to be signature or unique for the "classical" lineage, while three were unique for the "LLG/POS variant". The U277 (E. coli numbering) signature character, corresponding to a highly conserved residue of the 16S molecule, and the unique G681 residue, conserved in a functionally strategic region also of 16S, are the most pronounced attributes (autapomorphies) of the "classical" and the "LLG/POS variant" lineages, respectively. Both lineages were found to be descendants of a common ancestor with the Prk/Daruma C. psittaci variant. Compared with the "classical", the "LLG/POS variant" lineage has retained more ancestral features. The current rRNA secondary structure-based analysis and phylogenetic inference reveal new insights into how these two C. abortus lineages have differentiated during their evolution

Supplement (01-10)

Supplements described in manuscript: S1 is sequence input (fasta format); S2 is time-ordered list of species; S3 is CAST output; S4 is parsed BLAST output; S5 is MCL output; S6 is BLAST output for unique genes; S7 is genome distance matrix; S8 is full sequence dataset from Figure 4 (fasta format); S9 is sequence dataset from Figure 5 (fasta format); S10 is count of new family contributions

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available. (C) 2009 Elsevier B.V. All rights reserved

High-resolution melt PCR analysis for rapid identification of <em>Chlamydia abortus</em> live vaccine strain 1B among <em>C. abortus</em> strains and field isolates

International audienceWe describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP

Diversification and distribution of ruminant Chlamydia abortus clones assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolutionmode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations

The Chlamydiales Pangenome Revisited: Structural Stability and Functional Coherence

The entire publicly available set of 37 genome sequences from the bacterial order Chlamydiales has been subjected to comparative analysis in order to reveal the salient features of this pangenome and its evolutionary history. Over 2,000 protein families are detected across multiple species, with a distribution consistent to other studied pangenomes. Of these, there are 180 protein families with multiple members, 312 families with exactly 37 members corresponding to core genes, 428 families with peripheral genes with varying taxonomic distribution and finally 1,125 smaller families. The fact that, even for smaller genomes of Chlamydiales, core genes represent over a quarter of the average protein complement, signifies a certain degree of structural stability, given the wide range of phylogenetic relationships within the group. In addition, the propagation of a corpus of manually curated annotations within the discovered core families reveals key functional properties, reflecting a coherent repertoire of cellular capabilities for Chlamydiales. We further investigate over 2,000 genes without homologs in the pangenome and discover two new protein sequence domains. Our results, supported by the genome-based phylogeny for this group, are fully consistent with previous analyses and current knowledge, and point to future research directions towards a better understanding of the structural and functional properties of Chlamydiales

Data from: The Chlamydiales pangenome revisited: structural stability and functional coherence

The entire publicly available set of 37 genome sequences from the bacterial order Chlamydiales has been subjected to comparative analysis in order to reveal the salient features of this pangenome and its evolutionary history. Over 2,000 protein families are detected across multiple species, with a distribution consistent to other studied pangenomes. Of these, there are 180 protein families with multiple members, 312 families with exactly 37 members corresponding to core genes, 428 families with peripheral genes with varying taxonomic distribution and finally 1,125 smaller families. The fact that, even for smaller genomes of Chlamydiales, core genes represent over a quarter of the average protein complement, signifies a certain degree of structural stability, given the wide range of phylogenetic relationships within the group. In addition, the propagation of a corpus of manually curated annotations within the discovered core families reveals key functional properties, reflecting a coherent repertoire of cellular capabilities for Chlamydiales. We further investigate over 2,000 genes without homologs in the pangenome and discover two new protein sequence domains. Our results, supported by the genome-based phylogeny for this group, are fully consistent with previous analyses and current knowledge, and point to future research directions towards a better understanding of the structural and functional properties of Chlamydiales

Genome sequence of the Chlamydophila abortus variant strain LLG

Chlamydophila abortus is a common cause of ruminant abortion. Here we report the genome sequence of strain LLG, which differs genotypically and phenotypically from the wild-type strain S26/3. Genome sequencing revealed differences between LLG and S26/3 to occur in pseudogene content, in transmembrane head/inc family proteins, and in biotin biosynthesis genes

Minimum-spanning-tree analysis of 94 <i>C</i>. <i>abortus</i> isolates genotyped by MLST.

<p>Each circle represents a distinct MLST type (ST). The size of the circles is related to the number of strains, isolates or samples composing the particular STs. Solid lines connect single-locus variant STs while the light grey dashed line indicates the ST that differs in more than one locus; the number of locus variations is indicated between the circles. <b>A. Minimum-spanning-tree illustrating the evolutionary pathways of ruminant <i>C</i>. <i>abortus</i> clones.</b> The gray halo surrounding the circles delineates the <i>C</i>. <i>abortus</i> clonal complex. Alteration in MLST alleles compared with predicted ancestor ST19 are underlined. The circle colours indicate the corresponding MLVA types (MTs); nd, MT not determined; nt, MT not typeable. <b>B. Minimum-spanning-tree exemplifying the different ST distribution patterns among <i>C</i>. <i>abortus</i> strains, isolates and samples originating from France and Greece.</b> The colours indicate the corresponding countries of isolation (France, red; Greece, blue; Other countries, shades of gray). With asterisks (*) are labeled the field isolates or samples displaying the 1B-vaccine-type profile.</p

Dendrogram illustrating the relationships of MLST types (STs) on the basis of concatenated sequences of <i>gatA</i>, <i>hemN</i> and <i>fumC</i> loci, in comparison with other genotyping findings.

<p>The dendrogram includes 94 <i>C</i>. <i>abortus</i> strains and field isolates or samples, which were collected from ruminant in nine countries over a period of 60 years. The genotypic characteristics of clones are color-coded: the more distantly related ST30 is presented in blue whereas the other STs in shades of gray. The dendrogram was constructed by using the UPGMA algorithm. Linkage distances are indicated on the scale at the bottom. na, not available information; nd, not determined; nt, not typeable.</p