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

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

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 = 0.156, P = 0.784 for BBG; R = 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.Parts of this work were performed under the umbrella of the COST project (TD1303).Peer Reviewe

Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus

Ixodes ricinus transmits Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease. Previous studies have also detected Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis and several Babesia species in questing ticks in The Netherlands. In this study, we assessed the acarological risk of exposure to several tick-borne pathogens (TBPs), in The Netherlands.Questing ticks were collected monthly between 2006 and 2010 at 21 sites and between 2000 and 2009 at one other site. Nymphs and adults were analysed individually for the presence of TBPs using an array-approach. Collated data of this and previous studies were used to generate, for each pathogen, a presence/absence map and to further analyse their spatiotemporal variation.Rickettsia helvetica (31.1 %) and B. burgdorferi sensu lato (11.8 %) had the highest overall prevalence and were detected in all areas. Neoehrlichia mikurensis (5.6 %), A. phagocytophilum (0.8 %), and Babesia spp. (1.7 %) were detected in most, but not all areas. The prevalences of pathogens varied among the study areas from 0 to 64 %, while the density of questing ticks varied from 1 to 179/100 m2. Overall, 37 % of the ticks were infected with at least one pathogen and 6.3 % with more than one pathogen. 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. Alternatively, coinfection of R. helvetica with either B. afzelii or N. mikurensis occurred significantly less frequent.The diversity of TBPs 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

Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in natural rodent and tick communities in Southern Hungary.

The aim of this study was to investigate the natural cycle of the new human pathogenic bacteria Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in Southern Hungary. We collected rodents with live-traps (2010-2013) and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all the ectoparasites were removed and stored in 70% alcohol. We found relatively low overall prevalence of tick infestation (8%). Samples were analysed for A. phagocytophilum and Candidatus N. mikurensis with multiplex quantitative real-time PCR targeting a part of major surface protein 2 (msp2) and the heat shock protein groEL genes, respectively. The overall prevalence in tissue samples was 6.6% (skin) and 5.1% (spleen) for A. phagocytophilum and 1.7% (skin) and 3.4% (spleen) for Candidatus N. mikurensis. Candidatus N. mikurensis was only detected in Apodemus flavicollis and Apodemus agrarius, while A. phagocytophilum was found in A. flavicollis, A. agrarius, Myodes glareolus, Microtus arvalis and Mus musculus samples. Prevalence of A. phagocytophilum in skin samples of A. flavicollis was significantly higher than prevalence of N. mikurensis (p<0.05). Among questing Ixodes ricinus ticks we found three (8.8%) individuals (female, male, nymph) infected with Candidatus N. mikurensis. Five (3.1%) questing ticks had A. phagocytophilum infection (one I. ricinus male, two Dermacentor reticulatus females and two Haemaphysalis concinna females). We found one I. ricinus nymph removed from a male A. flavicollis with A. phagocytophilum infection. Our study provides new data on the occurrence of these pathogens in rodent tissue samples, questing ticks and engorged ticks in Southern Hungary

Spotted fever rickettsiae in wild-living rodents from south-western Poland.

Rickettsiae are obligate intracellular alpha-proteobacteria. They are transmitted via arthropod vectors, which transmit the bacteria between animals and occasionally to humans. So far, much research has been conducted to indicate reservoir hosts for these microorganisms, but our knowledge is still non-exhaustive. Therefore, this survey was undertaken to investigate the presence of Rickettsia spp. in wild-living small rodents from south-western Poland