Phylogeography and Genetic Diversity of Francisella tularensis subsp. holarctica in France (1947-2018)

In France, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. F. tularensis species presents low genetic diversity that remains poorly described in France, as only a few genomes of isolates from the country are available so far. The objective of this study was to characterize the genetic diversity of F. tularensis in France and describe the phylogenetic distribution of isolates through whole-genome sequencing and molecular typing. Whole genomes of 350 strains of human or animal origin, collected from 1947 to 2018 in France and neighboring countries, were sequenced. A preliminary classification using the established canonical single nucleotide polymorphism (canSNP) nomenclature was performed. All isolates from France (except four) belonged to clade B.44, previously described in Western Europe. To increase the resolution power, a whole-genome SNP analysis was carried out. We were able to accurately reconstruct the population structure according to the global phylogenetic framework, and highlight numerous novel subclades. Whole-genome SNP analysis identified 87 new canSNPs specific to these subclades, among which 82 belonged to clade B.44. Identifying genomic features that are specific to sublineages is highly relevant in epidemiology and public health. We highlighted a large number of clusters among a single clade (B.44), which shows for the first time some genetic diversity among F. tularensis isolates from France, and the star phylogeny observed in clade B.44-subclades revealed that F. tularensis biodiversity in the country is relatively recent and resulted from clonal expansion of a single population. No association between clades and hosts or clinical forms of the disease was detected, but spatiotemporal clusters were identified for the first time in France. This is consistent with the hypothesis of persistence of F. tularensis strains found in Western Europe in the environment, associated with slow replication rates. Moreover, the presence of identical genotypes across long periods of time, and across long distances, supports this hypothesis but also suggests long-distance dispersal of the bacterium.This work was supported by the French National Research Agency (ANR) and the Direction Générale de l’Armement (DGA) (No. ANR-15-ASTR-0021-01). MK is a Ph.D. student co-supported by Université Paris-Est and DGA grants

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe

International audienc

Co-circulation of different A. phagocytophilum variants within cattle herds and possible reservoir role for cattle

Background: Anaplasma phagocytophilum is a zoonotic tick-borne intracellular alpha-proteobacterium causing tick-borne fever, which leads to significant economic losses in domestic ruminants in Europe. Its epidemiological cycles are complex and reservoir host species of bovine strains have not yet been identified. Given that little genetic information is available on strains circulating within a defined bovine environment, our objective was to assess the genetic diversity of A. phagocytophilum obtained from the same farms over time. Methods: Blood samplings were performed several times in two European herds. In the French herd, 169 EDTA-blood samples were obtained from 115 cows (32 were sampled two to four times). In the German herd, 20 cows were sampled six times (120 EDTA-blood samples). The presence of A. phagocytophilum DNA was assessed using a qPCR targeting msp2. The positive DNA samples underwent MLST at nine genetic markers (typA, ctrA, msp4, pleD, recG, polA, groEL, gyrA, and ankA). For each locus, sequences were aligned with available bacterial sequences derived from cattle, horse, dog, and roe deer hosts, and concatenated neighbor joining trees were constructed using three to six loci. Results: Around 20% (57/289) of samples were positive. Forty positive samples from 23 French and six German cows (11 of them being positive at two time points) were sequenced. Six loci (typA, ctrA, msp4, pleD, recG, and polA) allowed to build concatenated phylogenetic trees, which led to two distinct groups of bovine variants in the French herd (hereafter called A and B), whereas only group A was detected in the German herd. In 42% of French samples, double chromatogram peaks were encountered in up to four loci. Eleven cows were found infected three weeks to 17 months after first sampling and harboured a new variant belonging to one or the other group. Conclusions: Our results demonstrate the occurrence of two major bovine strain groups and the simultaneous infection of single cows by more than one A. phagocytophilum strain. This challenges the role of cattle as reservoirs for A. phagocytophilum. This role may be facilitated via long-term bacterial persistence in individual cows and active circulation at the herd scale

Additional file 6: Figure S5. of Co-circulation of different A. phagocytophilum variants within cattle herds and possible reservoir role for cattle

NJ tree obtained using the concatenation of typA, ctrA, and msp4. Legends as in Fig. 1. (PDF 1438 kb

Additional file 1: Table S1. of Co-circulation of different A. phagocytophilum variants within cattle herds and possible reservoir role for cattle

DNA samples from Chastagner et al. [9] used for the alignments. The available sequences for each locus are indicated by an “X”. (XLSX 12 kb

Pragmàtica, 1772, 05, 29

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Additional file 4: Figure. S3. of Co-circulation of different A. phagocytophilum variants within cattle herds and possible reservoir role for cattle

NJ tree obtained using the concatenation of typA, ctrA, msp4, and recG. Legends as in Fig. 1. (PDF 1395 kb