A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus

Abstract

The efficacy of the mitochondrially encoded cytochrome b gene as a molecular marker for the discrimination of the reservoir host species of the Lyme borreliosis spirochete, Borrelia burgdorferi sensu lato (s.l.), in its European vector Ixodes ricinus (Acari: Ixodidae) was determined. Degenerate PCR primers were designed which amplified orthologous regions of the cytochrome b gene in several animal species which act as B. burgdorferi s.l. reservoirs and hosts for I. ricinus. PCR products were amplified and characterized by hybridization and restriction fragment length polymorphism analysis. Restriction fragment length polymorphism analysis of a 638-bp PCR product with HaeIII and DdeI revealed unique restriction fragment profiles, which allowed the taxonomic identification of animals to the genus level. A system was devised for the detection of the larval host blood meal from the remnants in unfed nymphal I. ricinus ticks by nested PCR amplification. An inverse correlation was demonstrated between amplicon size and successful PCR amplification of host DNA from the nymphal stage of the tick. The stability of the cytochrome b product as a marker for the identification of the larval host species in the nymphal instar was demonstrated up to 200 days after larval ingestion (approximately 165 days after molting) by reverse line blotting with a host-specific probe. This assay has the potential for the determination of the reservoir hosts of B. burgdorferi s.l. by using extracts from the same individual ticks for both the identification of the host species and the detection of the Lyme borreliosis spirochete.

Insights into the evolutionary history ofCervus(Cervidae, tribe Cervini) based on Bayesian analysis of mitochondrial marker sequences, with first indications for a new species

“…Complete cytb was amplified and sequenced in two separate segments using both newly designed primers and primers published previously: cytb483_F (this study) and cytb3 (Kirstein and Gray 1996) for one segment, cytb_cerv (this study) and cytoB-B2 (Ludt et al 2004) for the other. PCRs and sequencing of cytb were performed as for CR, but using 55°C as annealing temperature in the amplification profiles.…”

Ecology ofBorrelia burgdorferi sensu latoin Europe: transmission dynamics in multi-host systems, influence of molecular processes and effects of climate change

“…The first attempt to use this technology targeted the mitochondrial cytochrome b gene using a PCR followed by reverse line blot. It was possible to detect individual host species but generic primers for the PCR component did not cover a sufficient range of potential hosts (Kirstein & Gray, 1996). Targeting the 18S rRNA gene was more satisfactory for detection of blood from taxonomic groups of animals, but because this gene is not found in the mitochondria, there was a consequent loss of sensitivity for the detection of mammal blood compared with that from birds (Pichon et al, 2003.…”

Host identification in unfed ticks from stable isotope compositions (δ13C and δ15N)

“…Although it is possible to obtain some information by collecting feeding ticks directly from the hosts, unfed exophilic ixodid ticks are much more readily available because they can be collected from vegetation in large numbers by blanket-dragging or flagging. Such field-caught unfed ticks contain small remnants of the bloodmeal obtained by the previous stage and several attempts have been made to identify hosts by detecting their DNA (Kirstein & Gray, 1996;Pichon et al, 2005;Humair et al, 2007;Moran-Cadenas et al, 2007;Allan et al, 2010) or proteins (Vennestrom & Jensen, 2007;Wickramasekara et al, 2008;Laskay et al, 2012;Onder et al, 2013;Onder et al, 2014) in these bloodmeal remnants. These methods have met with some success, although they are limited by their complexity, expense and dwindling sensitivity as the bloodmeal degrades in the tick gut.…”