Genomic evidence that the live Chlamydia abortus vaccine strain 1B is not attenuated and has the potential to cause disease

Abstract

Background: The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis.

Methods: Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains).

Results: We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains.

Conclusions: The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated "reverted mutant" strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1-3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease.

Keywords: Chlamydia abortus; Genome analysis; Ovine enzootic abortion; Single nucleotide polymorphisms; Vaccination.

Fig. 1

Circular representation and genome comparison of C. abortus strains AB7, S26/3 and 1B/1H. The outer scale shows the size in base pairs. From the outside in: tracks 1 and 2 show the position of genes transcribed in a clockwise and anticlockwise direction, respectively. CDSs in tracks 1 and 2 are colour coded according to the function of their gene products: membrane or surface structures (dark green); central or intermediary metabolism (yellow); degradation of macromolecules (cyan); information transfer/cell division (red); degradation of small molecules (purple); regulators (pale blue); pathogenicity or adaptation (dark blue); energy metabolism (black); conserved hypothetical (orange); unknown (pale green); pseudogenes (brown). Track 3, SNP differences between AB7 and S26/3. Track 4, pseudogene similarities and differences between AB7 and S26/3: shared pseudogenes (brown); S26/3-specific pseudogenes (dark green); AB7-specific pseudogenes (red). Track 5, SNPs present in 1B relative to AB7: Burall et al. SNPs confirmed (magenta) or not identified (dark blue) in this study; Burall et al. SNPs in 1B relative to 1H which were not found in this study (cyan); unique SNP found in this study (dark green). Track 6, G + C content plot (in a 10 kb window); track 7, GC skew plot ([G − C]/[G + C] (in a 10 kb window). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Phylogenetic tree showing the relationship of strains within the vaccine strain clade using reference strain S26/3 as the outgroup. For clarity, the distance to S26/3 is not representative (as indicated by the dashed lines) and represents 604 SNPs from AB7. Ten SNPs separate AB7 from 1B-Cevac/1B-Enzovax with AB15, 11_669_5380/2 and 10DC0084 containing an additional 1, 2 and 3 SNPs, respectively.