Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Abstract

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 evolution mode. 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.

Fig 1. Dendrogram illustrating the relationships of MLST types (STs) on the basis of concatenated sequences of gatA, hemN and fumC loci, in comparison with other genotyping findings.

The dendrogram includes 94 C. abortus 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.

Fig 2. Minimum-spanning-tree analysis of 94 C. abortus isolates genotyped by MLST.

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. A. Minimum-spanning-tree illustrating the evolutionary pathways of ruminant C. abortus clones. The gray halo surrounding the circles delineates the C. abortus 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. Minimum-spanning-tree exemplifying the different ST distribution patterns among C. abortus strains, isolates and samples originating from France and Greece. 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.