Molecular Diagnosis of Emmonsia-Like Fungi Occurring in Wild Animals.

Using specific primers based on the ribosomal operon, positive DNA amplification was obtained from lungs of 11/215 tested small burrowing animals, both terrestrial and aquatic, and including frozen (n = 4) and formalin-fixed paraffin-embedded (n = 7) samples. The main species detected in Europe in mice, otters and river rats was Emmonsia crescens. Two strains from otters and weasels were Blastomyces parvus. Two Australian wombats revealed the presence of a hitherto unknown species of the geophilic genus Emmonsiellopsis.

The containment of potential outbreaks triggered by imported Chikungunya cases in Italy: a cost utility epidemiological assessment of vector control measures.

The arrival of infected travelers from endemic regions can trigger sustained autochthonous transmission of mosquito-borne pathogens in Europe. In 2007 a Chikungunya outbreak was observed in central Italy, mostly affecting two villages characterised by a high density of Aedes albopictus. The outbreak was mitigated through intervention strategies reducing the mosquito abundance. Ten years later, in 2017, sustained Chikungunya transmission was documented in both central and southern Italy. The proposed analysis identifies suitable reactive measures for the containment and mitigation of future epidemics by combining epidemiological modeling with a health economic approach, considering different arrival times of imported infections and possible delays in the notification of cases. Obtained estimates suggest that, if the first notification will occur in the middle of the mosquito breeding season, the combination of larvicides, adulticides and breeding sites removal represents the optimal strategy. In particular, we found that interventions implemented in 2007 were cost-effective, with about 3200 prevented cases, 1450 DALYs averted and €13.5 M saved. Moreover, larvicides are proven to be more cost beneficial in early summer and warmer seasons, while adulticides should be preferred in autumn and colder seasons. Our results provide useful indications supporting urgent decision-making of public health authorities in response to emerging mosquito-borne epidemics.

A 2-yr Mosquito Survey Focusing on Aedes koreicus (Diptera: Culicidae) in Northern Italy and Implications for Adult Trapping.

Aedes koreicus (Edwards) is an invasive mosquito species, like Aedes albopictus (Skuse) and Aedes japonicus japonicus (Theobald), that has already colonized a large part of northeastern Italy and other European countries. Despite its rapid expansion, information about adult distribution and trapping is lacking. Here, we conducted a 2-yr longitudinal survey using adult traps to investigate the spatiotemporal distribution of Ae. koreicus and evaluated the effectiveness of three trapping devices in Latin square experiments conducted in an urban site and a forested site. The following three different traps were compared: a CO2-baited Biogents (BG) Sentinel trap, a CO2-baited Centers for Disease Control and Prevention light trap (CDC trap), and a grass infusion-baited gravid trap.In northern Italy, Ae. koreicus was collected from late April to early November, with peak of abundance observed in August. Aedes koreicus was more abundant in 2015 than in 2014 because of higher temperatures during summer. Unlike Ae. albopictus, the abundance of Ae. koreicus was not related to the altitude of the sampling locations in the range 241-660 m above sea level. The BG Sentinel and gravid traps collected significantly more Ae. koreicus than the CDC trap in the urban site, whereas there was no significant difference between the three traps in the forested site. In the urban site, the BG Sentinel trap and the gravid trap were the most effective for collecting Ae. albopictus and Culex pipiens L., respectively. In the forested site, Cx. pipiens was primarily collected by the CDC trap.

Recent increase in prevalence of antibodies to Dobrava-Belgrade virus (DOBV) in yellow-necked mice in Northern Italy.

Dobrava-Belgrade virus (DOBV) is the most pathogenic hantavirus in Europe with a case-fatality rate of up to 12%. To detect changes in risk for humans, the prevalence of antibodies to DOBV has been monitored in a population of Apodemus flavicollis in the province of Trento (northern Italy) since 2000, and a sudden increase was observed in 2010. In the 13-year period of this study, 2077 animals were live-trapped and mean hantavirus seroprevalence was 2·7% (s.e. = 0·3%), ranging from 0% (in 2000, 2002 and 2003) to 12·5% (in 2012). Climatic (temperature and precipitation) and host (rodent population density, rodent weight and sex, and larval tick burden) variables were analysed using Generalized Linear Models and multi-model inference to select the best model. Climatic changes (mean annual precipitation and maximum temperature) and individual body mass had a positive effect on hantavirus seroprevalence. Other possible drivers affecting the observed pattern need to be studied further.

The challenge of West Nile virus in Europe: knowledge gaps and research priorities.

West Nile virus (WNV) is continuously spreading across Europe, and other continents, i.e. North and South America and many other regions of the world. Despite the overall sporadic nature of outbreaks with cases of West Nile neuroinvasive disease (WNND) in Europe, the spillover events have increased and the virus has been introduced into new areas. The high genetic diversity of the virus, with remarkable phenotypic variation, and its endemic circulation in several countries, require an intensification of the integrated and multidisciplinary research efforts built under the 7th Framework Programme of the European Union (FP7). It is important to better clarify several aspects of WNV circulation in Europe, including its ecology, genomic diversity, pathogenicity, transmissibility, diagnosis and control options, under different environmental and socio-economic scenarios. Identifying WNV endemic as well as infection-free areas is becoming a need for the development of human vaccines and therapeutics and the application of blood and organs safety regulations. This review, produced as a joint initiative among European experts and based on analysis of 118 scientific papers published between 2004 and 2014, provides the state of knowledge on WNV and highlights the existing knowledge and research gaps that need to be addressed with high priority in Europe and neighbouring countries.

Effects of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. I: empirical assessment.

Tick borne encephalitis (TBE) is endemic to eastern and central Europe with broad temporal and spatial variation in infection risk. Although many studies have focused on understanding the environmental and socio-economic factors affecting exposure of humans to TBE, comparatively little research has been devoted to assessing the underlying ecological mechanisms of TBE occurrence in enzootic cycles, and therefore TBE hazard. The aim of this study was to evaluate the effect of the main ungulate tick hosts on the pattern of tick infestation in rodents and TBE occurrence in rodents and questing adult ticks. In this empirical study, we considered three areas where endemic human TBE occurs and three control sites having no reported human TBE cases. In these six sites located in Italy and Slovakia, we assessed deer density using the pellet group count-plot sampling technique, collected questing ticks, live-trapped rodents (primarily Apodemus flavicollis and Myodes glareolus) and counted ticks feeding on rodents. Both rodents and questing ticks were screened for TBE infection. TBE infection in ticks and rodents was positively associated with the number of co-feeding ticks on rodents and negatively correlated with deer density. We hypothesise that the negative relationship between deer density and TBE occurrence on a local scale (defined by the minimum overlapping area of host species) could be attributed to deer (incompetent hosts) diverting questing ticks from rodents (competent hosts), know as the 'dilution effect hypothesis'. We observed that, after an initial increase, the number of ticks feeding on rodents reached a peak for an intermediate value of estimated deer density and then decreased. Therefore, while at a regional scale, tick host availability has already been shown to be directly correlated with TBE distribution, our results suggest that the interactions between deer, rodents and ticks are much more complex on a local scale, supporting the possibility of a dilution effect for TBE.

Effect of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. II: population and infection models.

Tick-borne encephalitis is an emerging vector-borne zoonotic disease reported in several European and Asiatic countries with complex transmission routes that involve various vertebrate host species other than a tick vector. Understanding and quantifying the contribution of the different hosts involved in the TBE virus cycle is crucial in estimating the threshold conditions for virus emergence and spread. Some hosts, such as rodents, act both as feeding hosts for ticks and reservoirs of the infection. Other species, such as deer, provide important sources of blood for feeding ticks but they do not support TBE virus transmission, acting instead as dead-end (i.e., incompetent) hosts. Here, we introduce an eco-epidemiological model to explore the dynamics of tick populations and TBE virus infection in relation to the density of two key hosts. In particular, our aim is to validate and interpret in a robust theoretical framework the empirical findings regarding the effect of deer density on tick infestation on rodents and thus TBE virus occurrence from selected European foci. Model results show hump-shaped relationships between deer density and both feeding ticks on rodents and the basic reproduction number for TBE virus. This suggests that deer may act as tick amplifiers, but may also divert tick bites from competent hosts, thus diluting pathogen transmission. However, our model shows that the mechanism responsible for the dilution effect is more complex than the simple reduction of tick burden on competent hosts. Indeed, while the number of feeding ticks on rodents may increase with deer density, the proportion of blood meals on competent compared with incompetent hosts may decrease, triggering a decline in infection. As a consequence, using simply the number of ticks per rodent as a predictor of TBE transmission potential could be misleading if competent hosts share habitats with incompetent hosts.

Saturation deficit and deer density affect questing activity and local abundance of Ixodes ricinus (Acari, Ixodidae) in Italy.

The wood tick Ixodes ricinus, one of the most common arthropod-borne disease vectors, is of increasing relevance for human and animal health in Europe. The aim of this study was to determine the relative contribution of several abiotic and biotic factors potentially affecting questing activity and local abundance of I. ricinus in Italy, considering the scale at which these factors interact with the host-seeking ticks. Within EDEN, a large-scale EU collaborative project on eco-epidemiology of vector-borne diseases, we collected questing ticks for three consecutive years using a standard protocol at eleven sites in the Italian Alps and Apennines. A total of 25 447 I. ricinus were collected. All sites showed the same annual pattern of tick activity (bimodal for nymphs and unimodal for larvae and adults), although the abundance of nymphs was statistically different between sites and years. A Generalized Linear Mixed Model and a Linear Mixed Model fitted to data for nymphs, showed that while the principal variables affecting the local abundance of questing ticks were saturation deficit (an index combining temperature and relative humidity) and red deer density, the most important variable affecting questing nymph activity was saturation deficit. As for the timing of seasonal emergence, we confirmed that the threshold temperature at this latitude for larvae is 10°C (mean maximum) while that for nymphs is 8°C.

The relative importance of host characteristics and co-infection in generating variation in Heligmosomoides polygyrus fecundity.

We examined the relative importance of intrinsic host factors and microparasite co-infection in generating variation in Heligmosomoides polygyrus fecundity, a parameter that serves as a proxy for infectiousness. We undertook extensive trapping of Apodemus flavicollis, the yellow-necked mouse in the woodlands of the Italian Alps and recorded eggs in utero from the dominant nematode species H. polygyrus, and tested for the presence of five microparasite infections. The results showed that sex and breeding status interact, such that males in breeding condition harboured more fecund nematodes than other hosts; in particular, worms from breeding males had, on average, 52% more eggs in utero than worms from non-breeding males. In contrast, we found a weak relationship between intensity and body mass, and no relationship between intensity and sex or intensity and breeding condition. We did not find any evidence to support the hypothesis that co-infection with microparasites contributed to variation in worm fecundity in this system. The age-intensity profiles for mice singly-infected with H. polygyrus and those co-infected with the nematode and at least one microparasite were both convex and not statistically different from each other. We concluded that intrinsic differences between hosts, specifically with regard to sex and breeding condition, contribute relatively more to the variation in worm fecundity than parasite co-infection status.

Heligmosomoides polygyrus reduces infestation of Ixodes ricinus in free-living yellow-necked mice, Apodemus flavicollis.

Free-living animals are usually inhabited by a community of parasitic species that can interact with each other and alter both host susceptibility and parasite transmission. In this study we tested the prediction that an increase in the gastrointestinal nematode Heligmosomoides polygyrus would increase the infestation of the tick Ixodes ricinus, in free-living yellow-necked mice, Apodemus flavicollis. An extensive cross-sectional trapping survey identified a negative relationship between H. polygyrus and I. ricinus counter to the prediction. An experimental reduction of the nematode infection through anthelmintic treatment resulted in an increase in tick infestation, suggesting that this negative association was one of cause and effect. Host characteristics (breeding condition and age) and habitat variables also contributed to affect tick infestation. While these results were counter to the prediction, they still support the hypothesis that interactions between parasite species can shape parasite community dynamics in natural systems. Laboratory models may act differently from natural populations and the mechanism generating the negative association is discussed.

Tick infestation on roe deer in relation to geographic and remotely sensed climatic variables in a tick-borne encephalitis endemic area.

Roe deer Capreolus capreolus are among the most important feeding hosts for the sheep tick Ixodes ricinus, thus contributing to the occurrence of tick-borne diseases in Europe. Tick-borne encephalitis (TBE), which is transmitted by co-feeding of larvae and nymphs on rodents, requires precise climatic conditions to occur. We used roe deer as sentinels for potential circulation of TBE virus in Northern Italy, by examining the association between tick infestation, occurrence of TBE human cases, geographical and climatic parameters. Tick infestation on roe deer, and particularly frequency of co-feeding, was clearly associated with the geographic location and the autumnal cooling rate. Consistently, TBE occurrence in humans was geographically related to co-feeding tick abundance. The surveillance of tick infestation on roe deer, combined with remotely sensed climatic data, could therefore be used as an inexpensive early risk assessment tool of favourable conditions for TBE emergence and persistence in humans.

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.

[Ixodes ricinus, transmitted diseases and reservoirs]. / Ixodes ricinus, malattie trasmesse e reservoirs.

The tick Ixodes ricinus has been recorded in most Italian regions especially in thermo-mesophilous woods and shrubby habitats where the relative humidity allow the tick to complete its 3 year developmental cycle, as predicted for the European climatic ranges. This tick acts both as vector and reservoir for a series of wildlife zoonotic pathogens, especially the agents of Lyme diseases, Tick borne encephalitis and Human Granulocytic Ehrlichiosis, which are emerging in most of Europe. To assess the spatial distribution of these pathogens and the infection risk for humans and animals within the territory of the Province of Trento, we carried out a long term study using a combination of eco-epidemiological surveys and mathematical modelling. An extensive tick collection with a GIS based habitat suitability analysis allowed us to identify the areas where tick occurs at various density. To identify the areas with higher infection risk, we estimated the values of R0 for Borrelia burgdorferi s.l., TBE virus and Anaplasma phagocytophila under different ecological conditions. We assessed the infection prevalence in the vector and in the wildlife reservoir species that play a central role in the persistence of these infections, ie the small mammals A. flavicollis and C. glareolus. We also considered the double effect of roe deer (Capreolus capreolus) which act as reservoir for A. phagocytophila but is an incompetent host for B. burgdorferi and TBE virus, thus reducing the infection prevalence in ticks of these last two pathogens. Infection prevalence with B. burgdorferi and A. phagocytophila in the vector was assessed by PCR screening 1212 I. ricinus nymphs collected by dragging in six main study areas during 2002. The mean infection prevalence recorded was 1.32% for B. burgdorferi s.l. and 9.84% for A. phagocytophila. Infection prevalence in nymphs with TBE virus, as assessed in a previous study was 0.03%. Infection prevalence in rodents was assessed by screening (with ELISA and PCR) tissues and blood samples collected from 367 rodent individuals trapped extensively during 2002 within 6 main study areas. A. flavicollis (N=238) was found to be infected with all three pathogens investigated, with infection prevalence ranging from 3.3% for TBE virus to 11.7% for A. phagocytophila, and 16.6% with B. burgdorferi s.l. C. glareolus (N=108) showed an infection prevalence of 6.5% with A. phagocytophila and 12.7% with B. burgdorferi s.l., while no individuals were infected with TBE virus. We also screened 98 spleen samples collected from roe deer with PCR, resulting in a mean prevalence of infection with A. phagocytophila of 19.8%. Using a deterministic model we explored the condition for diseases persistence under different rodent and roe deer densities. R0 values resulted largely above 1 for B. burgdorferi s.l. in the vast majority of the areas classified as suitable for I. ricinus occurrence in Trentino, while the condition for TBE persistence appeared to be more restricted by a combination of climatic condition and host densities.

Tick-borne encephalitis virus in northern Italy: molecular analysis, relationships with density and seasonal dynamics of Ixodes ricinus.

Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick-borne encephalitis (TBE)-specific antiserum. The identity of the virus isolates was determined by RT-PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of L. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.