The discovery of B. afzelii in England, its distribution and its origin
To date there have been a limited number of published studies concerning the infections found within tick populations in England and Wales. To the best of our knowledge the last conclusive study investigating LB infections in English questing ticks focused solely on a woodland in Southern England and found only B. garinii and B. valaisiana in questing ticks and in reservoir hosts (Kurtenbach et al., 1998). In Continental Europe field studies have indicated that B. afzelii is common and, in many cases, the dominant species (Etti et al., 2003; Rauter and Hartung, 2005;van Overbeek et al., 2008). In the Scottish highlands B. afzelii also appears to be the predominant species (Ling et al., 2000). It has been suggested previously that tick questing behaviour resulting in a lack of nymphal infestation of mice may prevent the existence of B. afzelii in England and Ireland (Gray et al., 1999; Randolph and Storey, 1999; Randolph et al., 1999), but it has also been a consideration that the lack of sites investigated in England and Wales make it difficult to speculate on.
Here we report B. afzelii in questing ticks from England. Just over half the sites were found to be supporting B. afzelii strains, and the MLSA scheme revealed that the infections were highly focal, with four of the five STs from England each found exclusively and often repeatedly at a single site. Although it is possible that isolated B. afzelii populations have been maintained in England for long periods of time, and may have diverged from each other by genetic drift, we consider it more likely that the genetically distinct localized populations represent independent and recent introductions from outside the UK. This view is supported by the PhyML tree, which reveals that the English STs are polyphyletic, hence there is no single common ancestor unique to English STs (Fig. 3). However, at present it is only possible to speculate on these strains' geographical origins. The goeBURST reveals that most English STs are not linked to the major clonal complex of the species (Fig. 2C), suggesting they have not recently spread from France, Germany or Latvia. The exception to this is ST164, which was observed at the Widcombe Hill site in England and is assigned as founder to three French STs in Fig. 2C. This infers a French origin of this strain, although the exact ST was not present in our sample from France. In general, the PhyML tree in Fig. 3 suggests that the English STs are more closely related to French and German STs than to the Latvian and Scottish STs.
Genomic regions with more genetic variation are required, such as whole genome intergenic SNP analysis, to allow for an estimate on the period since B. afzelii introduction to the English sites. Furthermore, a more extensive survey of European sites may reveal the origins of the English B. afzelii strains and also shed light on potential methods of entry for these rodent-associated strains.
Phylogeographic structuring of LB species
Host specialization is a key process in the ecology and evolution of tick-borne zoonotic diseases. In this study we aimed to further our understanding of the impact of host association on the spread of zoonotic vector-borne pathogens by analysing three species of the LB group of spirochetes that are specialized to either avian or rodent hosts. MLSA on housekeeping genes has revealed differences in the level of geographic structuring of populations of LB species that are consistent with patterns of migration of their different vertebrate hosts. Both bird-related species investigated, B. valaisiana and B. garinii, showed evidence of spatial mixing of STs between countries, while the rodent-related B. afzelii showed evidence of differentiation of populations from each of the four countries. This differentiation was pronounced to the extent that only two B. afzelii STs were found in more than one country.
This finding was statistically supported using an FST test for pairwise differentiation between populations. Interestingly, while in B. garinii we found no significant differentiation of populations in different countries, suggesting entirely free movement of strains, B. valaisiana showed low to moderate differentiation, suggesting there is not complete homogenization of B. valaisiana strains within Europe. This was surprising because both species appear to be transmitted by similar species of avian hosts (Taragel'ova et al., 2008; Dubska et al., 2009). However, our results suggest that subtle ecological differences may exist between these species and it has been known for a long time that, apart from transmission cycles in terrestrial birds, B. garinii populations are also maintained by seabirds and their associated tick, Ixodes uriae(Olsen et al., 1995; Bunikis et al., 1996; Larsson et al., 2007). It is interesting to note that Comstedt and colleagues (2009) reported an overlap of marine and terrestrial B. garinii populations, although whether or not this may play a role in the population structure observed in the present study remains speculative, since no B. garinii from marine transmission cycles have been analysed by MLSA to date.
Of the three species analysed in the present study, B. garinii was found to be the most diverse. The finding that B. garinii showed a higher genetic diversity than B. valaisiana has been reported previously and it has been proposed that this might be due to a more recent adaptation of B. garinii to avian hosts (Margos et al., 2009). This may also contribute to subtle ecological differences in host associations, and therefore to the differences in population patterns observed here. In the pairwise FST comparisons, the only population pair showing no significant differentiation in B. valaisiana was England/Latvia, which is the most geographically distant pair. Currently, it is not known whether the differentiation observed between the LB strains from France and England is due to bias created by the culturing of the LB strains from France. Further sampling of environmental LB strains from France would be required to confirm these findings.
The B. afzelii FST score was markedly higher than B. garinii or B. valaisiana, suggesting that movement of strains between countries is more restricted. In B. afzelii there is no significant trend to suggest that FST increases with geographical distance, which may implicate a role of geographical barriers in determining population structure. For example, it is likely that the English Channel acts as a barrier to the movement of B. afzelii strains between Great Britain and continental Europe.
The B. afzelii population in Latvia, where it is more common for the same ST to be found in different sites, is less clearly differentiated than in England, where different sites tend to be associated with specific genotypes. The three Scottish strains included in this study appear to be more closely related to Latvian STs than to English ones (Fig. 3), suggesting that there is limited, or potentially no, migration between north and south in the UK. This is interesting in light of a study by Searle and colleagues (2009) investigating three species of small mammals (including the field vole Microtus agrestis, bank vole Myodes glareolus and pygmy shrew Sorex minutus) in Great Britain. In each case, two geographically distinct phylogroups were found. All the species showed a clear north/south divide between the phylogroups, which occurred in northern England, in the case of M. agrestis, and as far north as central Scotland in the case of S. minutus. The marked differentiation between English and Scottish B. afzelii samples may therefore be a result of limited north–south rodent migration.
Taken together, the findings reported in this article support the hypothesis that the movement and spread of LB species is dependant on the host, not the vector, but further investigations on a finer genetic scale, and much denser sampling, are required to fully understand the subtle ecological differences between hosts and to reconstruct fully the migration patterns of the different LB species. Given that the migration of some LB species is limited by the propensity for their vertebrate hosts' ranges to shift, landscape genetic analysis would be an appropriate approach to determine barriers to migration (Manel et al., 2003). This is particularly important for understanding the migration and emergence patterns of B. afzelii, which is associated with rodent hosts. Such future investigations would be facilitated by (i) identification of the rodent hosts of B. afzelii in England and (ii) the use of neutral genetic markers.