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

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

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

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

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