Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

Tick-borne pathogens and their reservoir hosts in northern Italy.

The aim of this study was to determine the occurrence of Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., and Candidatus Neoehrlichia mikurensis in Ixodes spp. ticks removed from wildlife, domestic animals and humans in the Province of Trento (northern Italy) in order to better understand their ecology and provide public health professionals with an updated list of pathogens which should be considered during their diagnostic procedures after a tick bite. During 2011-2012, 848 feeding ticks at all life stages (adults, nymphs and larvae) from various hosts (wild ungulates, birds and rodents; domestic sheep, dogs and humans) were collected. The highest prevalences of A. phagocytophilum and Rickettsia spp. were detected in adult and nymphal tick stages feeding on wild ungulates (11.4% prevalence for both pathogens), while the Babesia spp. prevailed in nymphal and larval ticks feeding on wild birds (7.7%). A wide spectrum of tick-borne agents was present in larval ticks: those detached from wild ungulates were positive for A. phagocytophilum, B. venatorum, R. helvetica, R. monacensis and R. raoultii, while those removed from wild rodents were positive for B. venatorum, R. helvetica, R. monacensis and Ca. N. mikurensis, and ticks from wild birds carried A. phagocytophilum, B. venatorum, B. capreoli and R. helvetica. This study provides evidence of circulation of five tick-borne pathogens not reported in this region before, specifically R. raoultii, R. monacensis, B. venatorum, B. capreoli and B. microti. Furthermore, it discusses the epidemiological role of the animal species from which the ticks were collected highlighting the needs for more experimental studies especially for those pathogens where transovarial transmission in ticks has been demonstrated.

Identification of Ixodes ricinus blood meals using an automated protocol with high resolution melting analysis (HRMA) reveals the importance of domestic dogs as larval tick hosts in Italian alpine forests.

BACKGROUND: In Europe, Ixodes ricinus L. is the main vector of a variety of zoonotic pathogens, acquired through blood meals taken once per stage from a vertebrate host. Defining the main tick hosts in a given area is important for planning public health interventions; however, until recently, no robust molecular methods existed for blood meal identification from questing ticks. Here we improved the time- and cost-effectiveness of an HRMA protocol for blood meal analysis and used it to identify blood meal sources of sheep tick larvae from Italian alpine forests. METHODS: Nine hundred questing nymphs were collected using blanket-dragging in 18 extensive forests and 12 forest patches close to rural villages in the Province of Trento. Total DNA was either extracted manually, with the QIAamp DNA Investigator kit, or automatically using the KingFisher™ Flex Magnetic Particle Processors (KingFisher Cell and Tissue DNA Kit). Host DNA was amplified with six independent host group real-time PCR reactions and identified by means of HRMA. Statistical analyses were performed in R to assess the variables important for achieving successful identification and to compare host use in the two types of forest. RESULTS: Automating DNA extraction improved time- and cost-effectiveness of the HRMA protocol, but identification success fell to 22.4% (KingFisher™) from 55.1% (QIAamp), with larval hosts identified in 215 of 848 questing nymphs; 23 mixed blood meals were noted. However, the list of hosts targeted by our primer sets was extended, improving the potential of the method. Host identification to species or genus level was possible for 137 and 102 blood meals, respectively. The most common hosts were Rodentia (28.9%) and, unexpectedly, Carnivora (28.4%), with domestic dogs accounting for 21.3% of all larval blood meals. Overall, Cetartiodactyla species fed 17.2% of larvae. Passeriformes (14.6%) fed a significantly higher proportion of larvae in forest patches (22.3%) than in extensive forest (9.6%), while Soricomorpha (10.9%) were more important hosts in extensive forest (15.2%) than in forest patches (4.3%). CONCLUSIONS: The HRMA protocol for blood meal analysis is a valuable tool in the study of feeding ecology of sheep ticks, especially with the cost- and time- reductions introduced here. To our knowledge, we show for the first time that domestic dogs are important larval hosts in the Alps, which may have possible implications for tick-borne disease cycles in urbanized areas.

Identifying the last bloodmeal of questing sheep tick nymphs (Ixodes ricinus L.) using high resolution melting analysis.

The sheep tick, Ixodes ricinus L., is an important hematophagous vector of zoonotic disease of both veterinary and public health importance in Europe. Risk models for tick-borne diseases can be improved by identifying the main hosts of this species in any given area. However, this generalist tick stays on a host for only a few days a year over its life cycle, making the study of its feeding ecology difficult. In contrast, ticks can easily be collected from vegetation when they are questing. Molecular methods have proved to be a reliable alternative to field observation, but most current methods have low sensitivity and/or low identification success (i.e. hosts are only identified to taxonomic levels higher than species). In this study we use Real-time PCR coupled with High Resolution Melting Analysis (HRMA) to identify the source of the last bloodmeal in questing tick nymphs. Twenty of the most important tick hosts were grouped taxonomically and six group-specific primer sets, targeting short mitochondrial DNA regions, were designed de novo. Firstly, we show that these primers successfully amplify target host DNA (from host tissue or engorged ticks), and that HRMA can be used to reliably identify hosts to species (or genera in the case of Sorex and Apodemus). Secondly, the new protocol was tested on field-collected questing nymphs. Bloodmeal source was identified in 65.4% of 52 individuals. In 83.3% of these, the host was identified to species or genera using HRMA alone. Moreover, the primer sets designed here can unequivocally identify mixed bloodmeals. The combination of sensitivity and identification success together with the closed-tube and single step approach that minimizes contamination, make Real-time HRMA a good alternative to current methods for bloodmeal identification.