|Title||Maintenance of Borrelia burgdorferi s.l. diversity in enzootic cycles|
|Source||University. Promotor(en): Willem Takken; Herbert Prins, co-promotor(en): H. Sprong. - Wageningen : Wageningen University - ISBN 9789462579576 - 180|
PRI Bioint Diagnostics, Food Safety & Phytosanitary
|Publication type||Dissertation, internally prepared|
|Keyword(s)||borrelia burgdorferi - life cycle - tickborne diseases - metastigmata - ixodes ricinus - disease vectors - rodents - mammals - genetic analysis - multilocus sequence typing - lyme disease - levenscyclus - ziekten overgebracht door teken - vectoren, ziekten - knaagdieren - zoogdieren - genetische analyse - multi-locus sequentie typering - lyme-ziekte|
|Categories||Medical Entomology / Agricultural Acarology|
Lyme borreliosis is the most prevalent vector-borne disease in the temperate regions of the northern hemisphere. The bacteria that cause it are members of the Borrelia burgdorferi sensu lato complex, a group of spirochaetes which are transmitted by hard ticks of the Ixodes ricinus complex. In several European countries, including The Netherlands, the incidence of Lyme borreliosis has been on the rise for the last decades. The acarological risk of human infection with Borrelia burgdorferi s.l. has been defined as the density of infected questing nymphs. This definition assumes that the distribution of the various genospecies of Borrelia in Lyme borreliosis is reflected in their distribution in questing ticks; furthermore, it assumes that all Borrelia genospecies are considered equally hazardous for humans. In order to define effective intervention strategies for controlling the disease, more insight in the transmission dynamics of tick-borne pathogens, both between animals and ticks, but also from ticks to humans is needed. As part of a Dutch research programme – “Shooting the messenger” – this PhD thesis focussed on linking the transmission cycles of Lyme spirochaetes to the different clinical manifestations of Lyme borreliosis. To that end, I explored aspects of the ecology and molecular adaptations of B. burgdorferi s.l. at various scales, from complex to genospecies level.
The ecological adaptations of B. burgdorferi s.l. are underpinned by a complex genomic structure and gene expression, with large genetic variation among and within the genospecies. In Chapter 3, we prove that the 5S-23S rDNA intergenic spacer (IGS) is a suitable molecular marker for identification of B. burgdorferi s.l. to genospecies level, but also to characterize the genetic diversity at intragenospecies level and to detect genetic differentiation among the subpopulations of Borrelia. Consequently, we used this marker in combination with other genetic markers, in the studies addressing the genetic diversity of Borrelia in small mammals and humans.
The main transmission route of these bacteria is the interstadial one, from larvae to nymphs and from nymphs to adult ticks. Larvae of I. ricinus can become infected during a blood meal from an infected host and during a blood meal in the vicinity of an infected nymph feeding on a host, a process known as co-feeding. The infected engorged larvae then moult into infected nymphs, which can transmit the spirochaetes to new hosts. The same process is repeated in the next developmental stage – nymph to adult. Thus, the maintenance of the bacteria in enzootic cycles is dependent on various species of vertebrates and the ticks that feed on them. In order to identify the main vertebrate hosts responsible for the maintenance of B. burgdorferi in enzootic cycles, but also for feeding I. ricinus ticks, we conducted a meta-analysis on literature data (Chapter 2). Our quantification method suggests that only a few host species, which are amongst the most widespread species in the environment (rodents, thrushes and deer), feed the majority of I. ricinus individuals and that rodents infect the majority of I. ricinus larvae with B. burgdorferi s.l.. The increase in distribution and abundance of these species, could be one of the main causes for the increase in Lyme borreliosis incidence in Europe in recent decades.
While at genospecies level, there is host specificity, with B. afzelii associated with rodents and B. garinii with birds, we wanted to see if the same holds true at intragenospecies level, for the various genotypes of Borrelia. Chapter 4 focuses on the rodents, which were identified in the literature meta-analysis as being the main hosts for I. ricinus larvae as well as for Borrelia afzelii. We tested the multiple niche polymorphisms hypothesis, using IGS, dbpA and ospC as molecular markers for typing B. afzelii genotypes in fed larvae collected from rodents in various areas in The Netherlands. Despite the high genetic diversity within B. afzelii, there was no difference between wood mice and bank voles in the number and types of B. afzelii haplotypes they transmit. Additionally, we compared the quantitative role of bank voles and wood mice in B. afzelii and Neoehrlichia mikurensis maintenance, another emerging tick-borne pathogen in Europe. Neoehrlichia mikurensis prevalence was positively associated with B. afzelii. Mathematical models including tick burden and infection prevalence indicated that bank voles are better amplifiers of these two bacteria than wood mice. Our study suggests that wood mice and bank voles differ in their contribution to the dynamics of B. afzelii, and possibly other TBP, in questing ticks but not in their contribution to the genetic diversity of these microorganisms.
The density of the vertebrate hosts and the feeding preferences of the ticks should determine the prevalence of B. burgdorferi s.l. genospecies in questing ticks. We address this topic in Chapter 5, by testing 5,570 questing I. ricinus nymphs from 22 different areas in The Netherlands. We found an overall prevalence of 11.8% for B. burgdorferi s.l., with large and consistent variations among the various locations. As expected based on the results of Chapter 2, Borrelia afzelii was predominant (6.7 % of the questing ticks) among the B. burgdorferi s.l. genospecies. It was followed by B. garinii/B. bavariensis (1.5 %), B. valaisiana (1.2 %), and B. burgdorferi sensu stricto (0.2 %). We noticed that, over the usual range of questing ticks’ densities, the density of infected ticks is increasing with the overall density of questing ticks, and a downward trend might be observed only for questing tick densities of over 200/100 m2. This indicates that the density of questing nymphs is the main driver of the acarological risk of human exposure to B. burgdorferi s.l.
We also screened for the presence of other tick-borne pathogens that have previously been detected in questing ticks in The Netherlands: Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis and several Babesia spp. (Chapter 5). To test whether these pathogens might share similar enzootic cycles we looked for patterns of coinfection and seasonal dynamics of infection in questing I. ricinus nymphs. One-third of the Borrelia-positive ticks were infected with at least one other pathogen. Coinfection of B. afzelii with N. mikurensis and with Babesia spp. occurred significantly more often than single infections, indicating the existence of mutual reservoir hosts. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of their coinfections with B. burgdorferi s.l. underline the need to consider them when evaluating the risks of infection and subsequently the risk of disease following a tick bite.
Chapter 6 addresses the pathogenicity of B. burgdorferi s.l. genospecies and genotypes for humans, using the eight multilocus sequence typing scheme housekeeping genes (MLST) and IGS as molecular markers. The frequency of the Borrelia spp. in humans is compared to the frequency in questing ticks to assess the infectivity of the various genospecies and genotypes. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). Just as in questing ticks, B. afzelii was the most prevalent Borrelia in in human Lyme borreliosis. Borrelia afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. Despite its high incidence in ticks and erythema migrans, in terms of disease burden (as measured by disability-adjusted life year), B. afzelii is of least concern for public health. Other Borrelia spirochaetes that are rarely found in questing I. ricinus ticks, such as B. bavariensis, seem to be responsible for most of the neuroborreliosis cases – a more severe clinical symptom of Lyme borreliosis. This implies that the prevalence of B. burgdorferi s.l. in questing ticks does not necessarily reflect the incidence of human Lyme borreliosis. We found six multilocus sequence types that were significantly associated with clinical manifestations in humans and five IGS haplotypes that were associated with the human Lyme borreliosis cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs.
In this thesis, I have identified the most important vertebrate hosts for maintenance of B. burgdorferi s.l. in enzootic cycles. I have also shown that their density is reflected in the prevalence of B. burgdorferi s.l. in questing ticks. The comparative study of questing ticks and Lyme borreliosis indicated that some of the Borrelia genospecies have similar prevalences in the two sources. The findings in my thesis indicate, thus, that there is a link between the density of suitable hosts for ticks and Borrelia spp., the density of infected ticks and the distribution of the B. burgdorferi s.l. genospecies in Lyme borreliosis. There are exceptions, however, that cannot be explained by this simple thread line. Such a situation is the perceived association of B. bavariensis with rodents that is not reflected by its extremely low prevalence in questing ticks. Furthermore, this low prevalence cannot explain the overrepresentation of B. bavariensis in Lyme borreliosis. As result of the study of pathogenicity of the various Borrelia genospecies and genotypes, I suggest the separate hazard assignment for the Borrelia genospecies; this, in combination with the exposure (prevalence in questing ticks) would allow for individual genospecies/genotypes risk assessment. The findings in this thesis stress the importance of both ecological and clinical studies for addressing the public health issue of Lyme borreliosis.