Biology subjects

Fingerle, V.

Publications and source records attributed to Fingerle, V..

5 recordsLinked to original sources

bioRxiv2025

Fast and efficient Borrelia genome recovery from tick samples using Whole-Genome Amplification

Borrelia burgdorferi sensu lato bacteria are the causative agents of Lyme borreliosis, a multisystemic illness with an expanding epidemiology in temperate areas. Genomic studies on Borrelia are hindered by the difficulty of culturing procedures: standard protocols require extended incubation, substantial technical expertise, and are prone to failure, limiting timely recovery of isolates. This contributes to a low number of complete Borrelia genomes available in public repositories, particularly for species other than B. burgdorferi sensu stricto. Here we introduce a novel approach that overcomes the necessity for extended culture by utilizing Whole Genome Amplification (WGA) directly on freshly collected ticks, and that can be performed in parallel to classical culturing. The protocol is paired with a tailored bioinformatic pipeline designed to ensure accurate assembly and reliable downstream analyses. Benchmarking on multiple control isolates demonstrated that the method yields high-quality chromosomal assemblies. To demonstrate practical applicability, we applied the protocol to freshly collected ticks, successfully generating five high-quality Borrelia chromosomes (two B. lusitaniae, two B. afzelii and one B. garinii). By generating sequencing-ready DNA in five days rather than months, our protocol greatly streamlines the process and minimizes the effort associated with traditional culture-based methods. This workflow will facilitate broader representation of understudied Borrelia species and support future epidemiological, ecological, and evolutionary investigations on this pathogen.

genomics↗

bioRxiv2023

Development and Validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato

Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l.. Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays. The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borrelia species. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays. Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions and limits of detection, thereby limiting result ambiguities. HighlightsFour qPCR assays used in duplex were developed to detect Borrelia burgdorferi sensu lato. The limits of detection and quantification were defined according to state of the art standards. The specifications allow to detect B. burgdorferi sensu lato from different sampling sources.

molecular biology↗

bioRxiv2023

German Ixodes inopinatus samples may not actually represent this tick species

Ticks are important vectors of human and animal pathogens, but many questions remain unanswered regarding their taxonomy. Molecular sequencing methods have allowed research to start understanding the evolutionary history of even closely related tick species. Ixodes inopinatus is considered a sister species and highly similar to Ixodes ricinus, an important vector of many tick-borne pathogens in Europe, but identification between these species remains ambiguous with disagreement on the geographic extent of I. inopinatus. In 2018-2019, 1583 ticks were collected from breeding great tits (Parus major) in southern Germany, of which 45 were later morphologically identified as I. inopinatus. We aimed to confirm morphological identification using molecular tools. Utilizing two genetic markers (16S rRNA, TROSPA) (n=37) and whole genome sequencing of specific ticks (n=8), we were able to determine that German samples morphologically identified as I. inopinatus, genetically represent I. ricinus regardless of previous morphological identification and most likely are not I. ricinus/I. inopinatus hybrids. Further, our results showed that the entire mitochondrial genome, let alone singular mitochondrial genes (i.e., 16S), is unable to distinguish between I. ricinus and I. inopinatus. As most examples of I. inopinatus in Germany were based on morphology and mitochondrial sequences, the results of the current study brings into question whether I. inopinatus was properly identified in previous research and if this species exists in Central Europe. Our results highlight the power of utilizing genomic data in answering questions regarding tick taxonomy even when closely related species are considered. HighlightsO_LIGerman Ixodes inopinatus samples represent I. ricinus based on genomic data C_LIO_LIThe mitochondrial genome is not sufficient for delineation of I. inopinatus and I. ricinus C_LIO_LIGerman samples most likely do not represent I. ricinus/I. inopinatus hybrids C_LI

evolutionary biology↗

bioRxiv2022

Phylogenomic diversity elucidates mechanistic insights into Lyme borreliae host association

Host association- the selective adaptation of pathogens to specific host species - evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi (Bb), the main LD pathogen in North America - a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCEHost association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD) causing agent, B. burgdorferi (Bb), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC, with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction.

microbiology↗

bioRxiv2020

Tick infestation probability increases with human recreational pressure and exploratory behaviour in wild great tits (Parus major)

Ticks are parasites that feed on the blood of various vertebrate hosts, including many species of bird. Birds can disperse ticks over short and long distances, therefore impacting tick population dynamics. The likelihood that birds attract ticks should depend on their behaviour and the environment. We studied various key ecological variables (breeding density, human disturbance) and phenotypic traits (exploratory behaviour; body condition) proposed to predict tick burden in great tits (Parus major). Our study spanned over three years and 12 human-recreated plots, equipped with nest-boxes in southern Germany. Adult breeders were assessed for exploratory behaviour, tick burden, and body condition. For each plot, human disturbance was quantified as a human recreational pressure index during biweekly nest box inspections by scoring the number of recreants using the plots. Infestation probability but not tick burden increased with exploratory behaviour. We also found moderate support for a positive effect of recreational pressure on infestation probability. Further, body condition negatively predicted tick burden. Individuals were repeatable in tick burden across years. Our study implies that infestation probability and tick burden are governed by distinct ecological and phenotypic drivers. Our findings also highlight the importance of incorporating ecological and individual variation in host phenotypes to predict spatiotemporal distributions of ticks in nature. (207/250-word limit) Lay SummaryTicks use many birds as hosts, but why do some individuals have more or fewer ticks? Using a data collected over three years on great tit adults inhabiting 12 different nest-box plots, we showed that more explorative birds and those in highly recreated habitats were more likely to be infested with ticks. Exploratory behaviour and human disturbance could modify great tit habitat choice and, therefore, impact how often a birds and ticks encounter each other.

ecology↗