Molecular epidemiology of Coxiella burnetii in French livestock reveals the existence of three main genotype clusters and suggests species-specific associations as well as regional stability.

Q fever is a worldwide zoonosis caused by the bacterium Coxiella burnetii. In domestic ruminants, Q fever main clinical manifestations are abortions. Although the clinical signs may differ between ruminant species, C. burnetii's genetic diversity remains understudied in enzootic areas. Here, we focused on France, where Q fever is enzootic, with the aims to (a) identify potential associations between C. burnetii genotypes and ruminant host species; (b) assess the distribution of C. burnetii genotypes both within French farms and across France's major livestock-farming regions; and (c) suggest a subset of markers for future genotypic studies. We used DNA samples collected between 2006 and 2015 from 301 females (160 cows, 76 ewes, 65 goats) aborted of Q fever within 7 different farming regions. C. burnetii diversity was determined using a multiple-locus variable-number of tandem repeat analysis (MLVA) considering 17 markers. Using a phylogenetic approach, we identified 3 main genotypic clusters divided into 12 sub-clusters. These clusters were significantly associated with ruminant species: almost all the cattle genotypes were found in a "cattle-specific" cluster whereas small ruminants genotypes essentially grouped into the two other clusters. The clusters also proved stable over space and time, some genotypes being more specifically observed in certain farming regions. We also observed some within-farm diversity but this diversity was restricted to a same genotypic cluster. Finally, we identified 6 MLVA markers that maximized the representativeness of the diversity described. Overall, we highlighted that molecular epidemiology is a relevant approach to assess C. burnetii's genetic diversity and to reveal the existence of species-specific associations and regional stability. These results will be valuable in the field to trace genotype circulation among ruminants and from ruminants to humans. Ultimately, the potential links between genotypes and virulence traits need to be investigated to adapt control measures in livestock farms.

Validation study for using lab-on-chip technology for Coxiella burnetii multi-locus-VNTR-analysis (MLVA) typing: application for studying genotypic diversity of strains from domestic ruminants in France.

Coxiella burnetii, the etiologic bacterium of Q fever zoonosis, is still difficult to control. Ruminants are often carriers and involved in human epidemics. MLVA is a promising genotyping method for molecular epidemiology. Different techniques are used to resolve the MLVA band profiles such as electrophoresis on agarose gels, capillary electrophoresis or using the microfluidic Lab-on-Chip system. In this study, system based on microfluidics electrophoresis with Lab-on-Chip technology was assessed and applied on DNA field samples to investigate the genotypic diversity of C. burnetii strains circulating in France. The Lab-on-Chip technology was first compared to agarose gel electrophoresis. Subsequently, the set-up Lab-on-Chip technology was applied on 97 samples collected from ruminants in France using the 17 markers previously described. A discordance rate of 27% was observed between Lab-on-Chip and agarose gel electrophoresis. These discrepancies were checked and resolved by sequencing. The cluster analysis revealed classification based on host species and/or geographic origin criteria. Moreover, the circulation of different genotypic strains within the same farm was also observed. In this study, MLVA with Lab-on-Chip technology was shown to be more accurate, reproducible, user friendly and safer than gel electrophoresis. It also provides an extended data set from French ruminant C. burnetii circulating strains useful for epidemiological investigations. Finally, it raises some questions regarding the standardization and harmonization of C. burnetii MLVA genotyping.

Impact of IS1111 insertion on the MLVA genotyping of Coxiella burnetii.

Q fever epidemiological investigations of the likely sources of contamination may involve Coxiella burnetii MLVA for direct and rapid typing from clinical samples. However, little information is available with regards to PCR amplification failures in C. burnetii MLVA typing. This paper focuses on difficulties encountered with MLVA loci that may impact the interpretation of MLVA data and shows that some loci may constitute hotspots for mutational events. MLVA genotyping, using 17 different loci, was used on vaginal swabs (VS) from clinically infected animals as described elsewhere (Chmielewski et al., 2009). Amplicons of interest were sequenced and identified using the BLAST software by comparison with sequences available in GenBank. All VS samples produced MLVA patterns. However, amplification failures or unexpected sizes amplicons (>to 1.5 kbp), making the interpretation of MLVA complicated, were also observed. Sequencing of these amplicons revealed the presence of IS1111 element insertion. In this C. burnetii MLVA study some difficulties encountered with genotyping are highlighted and the role of IS1111 element in genome plasticity is confirmed. Finally, the need for the selection of a set of VNTRs for an efficient MLVA scheme and the question of standardization and harmonization for comparable MLVA typing data are raised again.