Valentina Tagliapietra

Forest Structure and Roe Deer Abundance Predict Tick-Borne Encephalitis Risk in Italy

Background The Western Tick-borne encephalitis (TBE) virus often causes devastating or lethal disease. In Europe, the number of human TBE cases has increased dramatically over the last decade, risk areas are expanding and new foci are being discovered every year. The early localisation of new TBE foci and the identification of the main risk factors associated with disease emergence represent a priority for the public health community. Although a number of socio-economic parameters have been suggested to explain TBE upsurges in eastern Europe, the principal driving factors in relatively stable western European countries have not been identified. Methodology/Principal Findings In this paper, we analyse the correlation between the upsurge of TBE in 17 alpine provinces in northern Italy from 1992 to 2006 with climatic variables, forest structure (as a proxy for small mammal reservoir host abundance), and abundance of the principal large vertebrate tick host (roe deer), using datasets available for the last 40 years. No significant differences between the pattern of changes in climatic variables in provinces where TBE has emerged compared to provinces were no clinical TBE cases have been observed to date. Instead, the best model for explaining the increase in TBE incidence in humans in this area include changes in forest structure, in particular the ratio of coppice to high stand forest, and the density of roe deer. Conclusion/Significance Substantial changes in vegetation structure that improve habitat suitability for the main TBE reservoir hosts (small mammals), as well as an increase in roe deer abundance due to changes in land and wildlife management practices, are likely to be among the most crucial factors affecting the circulation potential of Western TBE virus and, consequently, the risk of TBE emergence in humans in western Europe. We believe our approach will be useful in predicting TBE risk on a wider scale.

Localized deer absence leads to tick amplification

Deer support high tick intensities, perpetuating tick populations, but they do not support tick-borne pathogen transmission, so are dilution hosts. We test the hypothesis that absence of deer (loss of a dilution host) will result in either an increase or a reduction in tick density, and that the outcome is scale dependent. We use a complementary methodological approach starting with meta-analysis, followed up by a field experiment. Meta-analysis indicated that larger deer exclosures reduce questing (host-seeking) tick density, but as the exclosure becomes smaller (<2.5 ha) the questing tick density is increased (amplified). To determine the consequences for tick-borne pathogen transmission we carried out a field experiment, comparing the intensity of ticks that fed on hosts competent for tickborne pathogen transmission (rodents) in two small (<1 ha) deer exclosures and their replicated controls. Intensity of larval ticks on rodents was not significantly different between treatments, but nymph intensity, the tick stage responsible for tick-borne encephalitis (TBE) transmission, was higher in deer exclosures. TBE seropositive rodents were found in a deer exclosure but not in the controls. We propose that localized absence of deer (loss of a dilution host) increases tick feeding on rodents, leading to the potential for tick-borne disease hotspots.

Hantavirus and arenavirus antibody prevalence in rodents and humans in Trentino, Northern Italy

The spatial and temporal distribution of hantavirus and arenavirus antibody-positive wild rodents in Trentino, Italy, was studied using immunofluorescence assays (IFA) in two long-term sites trapped in 2000-2003, and six other sites trapped in 2002. The overall hantavirus seroprevalence in the bank voles, Clethrionomys glareolus (n=229) screened for Puumala virus (PUUV) antibodies was 0.4%, and that for Apodemus flavicollis mice (n=1416) screened for Dobrava virus (DOBV) antibodies was 0.2%. Antibodies against lymphocytic choriomeningitis virus (LCMV) were found in 82 (5.6%) of the 1472 tested rodents; the seroprevalence being 6.1% in A. flavicollis (n=1181), 3.3% in C. glareolus (n=276), and 14.3% in Microtus arvalis (n=7). Of the serum samples of 488 forestry workers studied by IFA, 12 were LCMV-IgG positive (2.5%) and one DOBV-IgG positive (0.2%), however, the latter could not be confirmed DOBV-specific with a neutralization assay. Our results show a widespread distribution but low prevalence of DOBV in Trentino, and demonstrate that the arenavirus antibodies are a common finding in several other rodent species besides the house mouse.

Genetic and Ecologic Variability among Anaplasma phagocytophilum Strains, Northern Italy

To the Editor: The tick-borne pathogen Anaplasma phagocytophilum is an increasing potential public health threat across Europe. Its intraspecific genetic variability is associated with different reservoir host and vector tick species (1–4); however, the roles of various vertebrates as competent reservoirs of A. phagocytophilum in Europe need clarification (1). During March 2011–June 2013, we studied the prevalence and genetic variability of A. phagocytophilum in 821 questing Ixodes ricinus ticks (155 adults [A], 666 nymphs [N] collected by standard blanket dragging) and 284 engorged ixodid ticks (61A, 191N, 21 larvae [L]) collected from humans, dogs, sheep, hunted wild ungulates, live-trapped birds, and rodents. Blood samples from 1,295 rodents (yellow-necked mice [Apodemus flavicollis]), bank voles [Myodes glareolus], and harvest mice [Moscardinus avellanarius]) were also analyzed. All animal-handling procedures and ethical issues were approved by the Provincial Wildlife Management Committee (authorization n. 595 issued on 04.05.2011). The study site, Valle dei Laghi (northeastern Italian Alps), is a well-studied focus of emerging tick-borne pathogens in northern Italy (4).

Spatial and Temporal Dynamics of Lymphocytic Choriomeningitis Virus in Wild Rodents, Northern Italy

Prevalence of virus infection was higher in areas of greater rodent density.

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.

Pattern of Tick Aggregation on Mice: Larger Than Expected Distribution Tail Enhances the Spread of Tick-Borne Pathogens

The spread of tick-borne pathogens represents an important threat to human and animal health in many parts of Eurasia. Here, we analysed a 9-year time series of Ixodes ricinus ticks feeding on Apodemus flavicollis mice (main reservoir-competent host for tick-borne encephalitis, TBE) sampled in Trentino (Northern Italy). The tail of the distribution of the number of ticks per host was fitted by three theoretical distributions: Negative Binomial (NB), Poisson-LogNormal (PoiLN), and Power-Law (PL). The fit with theoretical distributions indicated that the tail of the tick infestation pattern on mice is better described by the PL distribution. Moreover, we found that the tail of the distribution significantly changes with seasonal variations in host abundance. In order to investigate the effect of different tails of tick distribution on the invasion of a non-systemically transmitted pathogen, we simulated the transmission of a TBE-like virus between susceptible and infective ticks using a stochastic model. Model simulations indicated different outcomes of disease spreading when considering different distribution laws of ticks among hosts. Specifically, we found that the epidemic threshold and the prevalence equilibria obtained in epidemiological simulations with PL distribution are a good approximation of those observed in simulations feed by the empirical distribution. Moreover, we also found that the epidemic threshold for disease invasion was lower when considering the seasonal variation of tick aggregation.

Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 20151

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

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

BackgroundIn 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.MethodsNine 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.ResultsAutomating 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%).ConclusionsThe 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.

Prevalence and genetic variability of Anaplasma phagocytophilum in wild rodents from the Italian alps

BackgroundHuman granulocytic anaplasmosis is a zoonotic bacterial disease with increasing relevance for public health in Europe. The understanding of its sylvatic cycle and identification of competent reservoir hosts are essential for improving disease risk models and planning preventative measures.ResultsIn 2012 we collected single ear biopsy punches from 964 live-trapped rodents in the Province of Trento, Italy. Genetic screening for Anaplasma phagocytophilum (AP) was carried out by PCR amplification of a fragment of the 16S rRNA gene. Fifty-two (5.4%) samples tested positive: 49/245 (20%) from the bank vole (Myodes glareolus) and 3/685 (0.4%) samples collected from the yellow-necked mouse (Apodemus flavicollis). From these 52 positive samples, we generated 38 groEL and 39 msp4 sequences. Phylogenetic analysis confirmed the existence of a distinct rodent strain of AP.ConclusionsOur results confirm the circulation of a specific strain of AP in rodents in our study area; moreover, they provide further evidence of the marginal role of A. flavicollis compared to M. glareolus as a reservoir host for this pathogen.