Maintenance of Borrelia burgdorferi s.l. diversity in enzootic cycles

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. </p

The genetic diversity of Borrelia afzelii is not maintained by the diversity of the rodent hosts.

Small mammals are essential in the enzootic cycle of many tick-borne pathogens (TBP). To understand their contribution to the genetic diversity of Borrelia afzelii, the most prevalent TBP in questing Ixodes ricinus, we compared the genetic variants of B. afzelii at three distinct genetic loci. We chose two plasmid loci, dbpA and ospC, and a chromosomal one, IGS

Nested coevolutionary networks shape the ecological relationships of ticks, hosts, and the Lyme disease bacteria of the Borrelia burgdorferi (s.l.) complex

Background: The bacteria of the Borrelia burgdorferi (s.l.) (BBG) complex constitute a group of tick-transmitted pathogens that are linked to many vertebrate and tick species. The ecological relationships between the pathogens, the ticks and the vertebrate carriers have not been analysed. The aim of this study was to quantitatively analyse these interactions by creating a network based on a large dataset of associations. Specifically, we examined the relative positions of partners in the network, the phylogenetic diversity of the tick''s hosts and its impact on BBG circulation. The secondary aim was to evaluate the segregation of BBG strains in different vectors and reservoirs. Results: BBG circulates through a nested recursive network of ticks and vertebrates that delineate closed clusters. Each cluster contains generalist ticks with high values of centrality as well as specialist ticks that originate nested sub-networks and that link secondary vertebrates to the cluster. These results highlighted the importance of host phylogenetic diversity for ticks in the circulation of BBG, as this diversity was correlated with high centrality values for the ticks. The ticks and BBG species in each cluster were not significantly associated with specific branches of the phylogeny of host genera (R 2 = 0.156, P = 0.784 for BBG; R 2 = 0.299, P = 0.699 for ticks). A few host genera had higher centrality values and thus higher importance for BBG circulation. However, the combined contribution of hosts with low centrality values could maintain active BBG foci. The results suggested that ticks do not share strains of BBG, which were highly segregated among sympatric species of ticks. Conclusions: We conclude that BBG circulation is supported by a highly redundant network. This network includes ticks with high centrality values and high host phylogenetic diversity as well as ticks with low centrality values. This promotes ecological sub-networks and reflects the high resilience of BBG circulation. The functional redundancy in BBG circulation reduces disturbances due to the removal of vertebrates as it allows ticks to fill other biotic niches

Eco-epidemiology of Borrelia miyamotoi and Lyme borreliosis spirochetes in a popular hunting and recreational forest area in Hungary

BACKGROUND: Borrelia miyamotoi, the newly discovered human pathogenic relapsing fever spirochete, and Borrelia burgdorferi sensu lato are maintained in natural rodent populations. The aim of this study was to investigate the natural cycle of B. miyamotoi and B. burgdorferi s.l. in a forest habitat with intensive hunting, forestry work and recreational activity in Southern Hungary. METHODS: We collected rodents with modified Sherman-traps during 2010–2013 and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all ectoparasites were removed and stored. Samples were screened for pathogens with multiplex quantitative real-time polymerase chain reaction (qPCR) targeting a part of flagellin gene, then analysed with conventional PCRs and sequencing. RESULTS: 177 spleen and 348 skin samples of six rodent species were individually analysed. Prevalence in rodent tissue samples was 0.2 % (skin) and 0.5 % (spleen) for B. miyamotoi and 6.6 % (skin) and 2.2 % (spleen) for B. burgdorferi s.l. Relapsing fever spirochetes were detected in Apodemus flavicollis males, B. burgdorferi s.l. in Apodemus spp. and Myodes glareolus samples. Borrelia miyamotoi was detected in one questing Ixodes ricinus nymph and B. burgdorferi s.l in nymphs and adults. In the ticks removed from rodents DNA amplification of both pathogens was successful from I. ricinus larvae (B. miyamotoi 5.6 %, B. burgdorferi s.l. 11.1 %) and one out of five nymphs while from Ixodes acuminatus larvae, and nymph only B. burgdorferi s.l. DNA was amplified. Sequencing revealed B. lusitaniae in a questing I. ricinus nymph and altogether 17 B. afzelii were identified in other samples. Two Dermacentor marginatus engorged larva pools originating from uninfected hosts were also infected with B. afzelii. CONCLUSIONS: This is the first report of B. miyamotoi occurrence in a natural population of A. flavicollis as well as in Hungary. We provide new data about circulation of B. burgdorferi s.l. in rodent and tick communities including the role of I. acuminatus ticks in the endophilic pathogen cycle. Our results highlight the possible risk of infection with relapsing fever and Lyme borreliosis spirochetes in forest habitats especially in the high-risk groups of hunters, forestry workers and hikers

Circulation of four Anaplasma phagocytophilum ecotypes in Europe.

BACKGROUND Anaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved. METHODS The presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses. RESULTS DNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion. CONCLUSION Four ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals

Comparing Multiple Locus Variable-Number Tandem Repeat Analyses with Whole-Genome Sequencing as Typing Method for Salmonella Enteritidis Surveillance in The Netherlands, January 2019 to March 2020

In the Netherlands, whole-genome sequencing (WGS) was implemented as routine typing tool for Salmonella Enteritidis isolates in 2019. Multiple locus variable-number tandem repeat analyses (MLVA) was performed in parallel. The objective was to determine the concordance of MLVA and WGS as typing methods for S. Enteritidis isolates. We included S. Enteritidis isolates from patients that were subtyped using MLVA and WGS-based core-genome Multilocus Sequence Typing (cgMLST) as part of the national laboratory surveillance of Salmonella during January 2019 to March 2020. The concordance of clustering based on MLVA and cgMLST, with a distance of ≤5 alleles, was assessed using the Fowlkes-Mallows (FM) index, and their discriminatory power using Simpson's diversity index. Of 439 isolates in total, 404 (92%) were typed as 32 clusters based on MLVA, with a median size of 4 isolates (range:2 to 141 isolates). Based on cgMLST, 313 (71%) isolates were typed as 48 clusters, with a median size of 3 isolates (range:2 to 39 isolates). The FM index was 0.34 on a scale from 0 to 1, where a higher value indicates greater similarity between the typing methods. The Simpson's diversity index of MLVA and cgMLST was 0.860 and 0.974, respectively. The median cgMLST distance between isolates with the same MLVA type was 27 alleles (interquartile range [IQR]:17 to 34 alleles), and 2 alleles within cgMLST clusters (IQR:1-5 alleles). This study shows the higher discriminatory power of WGS over MLVA and a poor concordance between both typing methods regarding clustering of S. Enteritidis isolates. IMPORTANCE Salmonella is the most frequently reported agent causing foodborne outbreaks and the second most common zoonoses in the European Union. The incidence of the most dominant serotype Enteritidis has increased in recent years. To differentiate between Salmonella isolates, traditional typing methods such as pulsed-field gel electrophoresis (PFGE) and multiple locus variable-number tandem repeat analyses (MLVA) are increasingly replaced with whole-genome sequencing (WGS). This study compared MLVA and WGS-based core-genome Multilocus Sequence Typing (cgMLST) as typing tools for S. Enteritidis isolates that were collected as part of the national Salmonella surveillance in the Netherlands. We found a higher discriminatory power of WGS-based cgMLST over MLVA, as well as a poor concordance between both typing methods regarding clustering of S. Enteritidis isolates. This is especially relevant for cluster delineation in outbreak investigations and confirmation of the outbreak source in trace-back investigations

Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts

Background: Lyme borreliosis is the most common tick-borne human disease and is caused by Borrelia burgdorferi sensu lato (s.l.). Borrelia miyamotoi, a relapsing fever spirochaete, is transmitted transovarially, whereas this has not been shown for B. burgdorferi (s.l). Therefore, B. burgdorferi (s.l) is considered to cycle from nymphs to larvae through vertebrates. Larvae of Ixodes ricinus are occasionally B. burgdorferi (s.l) infected, but their vector competence has never been studied. Methods: We challenged 20 laboratory mice with field-collected larvae of I. ricinus. A subset of these larvae was analysed for infections with B. burgdorferi (s.l) and B. miyamotoi. After three to four challenges, mice were sacrificed and skin and spleen samples were analysed for infection by PCR and culture. Results: Field-collected larvae were naturally infected with B. burgdorferi (s.l) (0.62 %) and B. miyamotoi (2.0 %). Two mice acquired a B. afzelii infection and four mice acquired a B. miyamotoi infection during the larval challenges. Conclusion: We showed that larvae of I. ricinus transmit B. afzelii and B. miyamotoi to rodents and calculated that rodents have a considerable chance of acquiring infections from larvae compared to nymphs. As a result, B. afzelii can cycle between larvae through rodents. Our findings further imply that larval bites on humans, which easily go unnoticed, can cause Lyme borreliosis and Borrelia miyamotoi disease.</p

New insights into the epidemiology of Listeria monocytogenes – A cross-sectoral retrospective genomic analysis in the Netherlands (2010–2020)

IntroductionListeriosis, caused by infection with Listeria monocytogenes (Lm), is a relatively rare but severe disease with one of the highest mortality rates among bacterial foodborne illnesses. A better understanding on the degree of Lm clustering, the temporal distribution of the clusters, and their association with the various food sources is expected to lead to improved source tracing and risk-based sampling.MethodsWe investigated the genomic epidemiology of Lm in the Netherlands between 2010 and 2020 by analyzing whole-genome-sequencing (WGS) data of isolates from listerioss patients and food sources from nationwide integrated surveillance and monitoring. WGS data of 756 patient and 770 food/environmental isolates was assessed using core-genome multi-locus sequence typing (cgMLST) with Hamming distance as measure for pairwise distances. Associations of genotype with the epidemiological variables such as patient’s age and gender, and systematic use of specific drugs were tested by multinomial logistic regressions. Genetic differentiation of the Lm within and between food categories was calculated based on allele frequencies at the 1701 cgMLST loci in each food category.ResultsWe confirmed previous results that some clonal complexes (CCs) are overrepresented among clinical isolates but could not identify any epidemiological risk factors. The main findings of this study include the observation of a very weak attribution of Lm types to food categories and a much better attribution to the producer level. In addition, we identified a high degree of temporal persistence of food, patient and mixed clusters, with more than half of the clusters spanning over more than 1 year and up to 10  years.DiscussionTaken together this would indicate that identifying persistent contamination in food production settings, and producers that process a wide variety of raw food produce, could significantly contribute to lowering the Lm disease burden