Annapaola Rizzoli

Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks.

Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geographic regions in Northern Italy by means of V6-16S rRNA amplicon pyrosequencing and community based ecological analysis. A total of 108 genera belonging to representatives of all bacterial phyla were detected and a rapid qualitative assessment for pathogenic bacteria, such as Borrelia, Rickettsia and Candidatus Neoehrlichia, and for other bacteria with mutualistic relationship or undetermined function, such as Wolbachia and Rickettsiella, was possible. Interestingly, the ecological analysis revealed that the bacterial community structure differed between the examined geographic regions and tick life stages. This finding suggests that the environmental context (abiotic and biotic factors) and host-selection behaviors affect their microbiome. Our data provide the most complete picture to date of the bacterial communities present within I. ricinus under natural conditions by using high-throughput sequencing technologies. This study further demonstrates a novel detection strategy for the microbiomes of arthropod vectors in the context of epidemiological and ecological studies.

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.

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.

Climatic factors driving invasion of the tiger mosquito (Aedes albopictus) into new areas of Trentino, northern Italy.

Background The tiger mosquito (Aedes albopictus), vector of several emerging diseases, is expanding into more northerly latitudes as well as into higher altitudes in northern Italy. Changes in the pattern of distribution of the tiger mosquito may affect the potential spread of infectious diseases transmitted by this species in Europe. Therefore, predicting suitable areas of future establishment and spread is essential for planning early prevention and control strategies. Methodology/Principal Findings To identify the areas currently most suitable for the occurrence of the tiger mosquito in the Province of Trento, we combined field entomological observations with analyses of satellite temperature data (MODIS Land Surface Temperature: LST) and human population data. We determine threshold conditions for the survival of overwintering eggs and for adult survival using both January mean temperatures and annual mean temperatures. We show that the 0°C LST threshold for January mean temperatures and the 11°C threshold for annual mean temperatures provide the best predictors for identifying the areas that could potentially support populations of this mosquito. In fact, human population density and distance to human settlements appear to be less important variables affecting mosquito distribution in this area. Finally, we evaluated the future establishment and spread of this species in relation to predicted climate warming by considering the A2 scenario for 2050 statistically downscaled at regional level in which winter and annual temperatures increase by 1.5 and 1°C, respectively. Conclusions/Significance MODIS satellite LST data are useful for accurately predicting potential areas of tiger mosquito distribution and for revealing the range limits of this species in mountainous areas, predictions which could be extended to an European scale. We show that the observed trend of increasing temperatures due to climate change could facilitate further invasion of Ae. albopictus into new areas.

First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy

Ixodes ricinus from Italy were examined for the first time to detect whether rickettsiae were present. Using molecular methods, we detected three different spotted fever group rickettsiae, including Rickettsia helvetica. Our results raise the possibility that bacteria other than R. conorii are involved in rickettsial diseases in Italy.

Detection of novel insect flavivirus sequences integrated in Aedes albopictus (Diptera: Culicidae) in Northern Italy.

The presence of DNA sequences integrated from a new flavivirus related to Cell Fusing Agent and Kamiti River Virus was identified in wild Aedes albopictus mosquito populations from the provinces of Trentino and Padova, Northern Italy. Field work was developed during August–October 2007 with BG-traps, and mosquitoes were screened for flavivirus and alphavirus. No alphavirus was detected, indicating that Chikungunya virus is not present in these mosquitoes in Trentino and Padova area. However, 21% of the pools were positive for flavivirus, further recognised with BLAST as similar to Kamiti River Virus. Phylogenetical analysis with 708 nucleotides from the NS5 gene identified this virus as a new member of the insect flavivirus clade, together with others like Kamiti River Virus, Cell Fusing Agent or Culex flavivirus, and in the group of those transmitted by Aedes. Furthermore, the treatment with RNAse, indicated that this flavivirus should be integrated in the genome of Ae. albopictus. These results propose that these sequences are transmitted by both sexes, and with different prevalence in the studied populations, and support the idea of a widespread distribution of integrated genomes in several mosquitoes from different areas, as first demonstrated with Cell Silent Agent. Evolutionary implications of this discovery and application in flavivirus phylogeny are discussed.

Effects of temperature and rainfall on the activity and dynamics of host-seeking Aedes albopictus females in northern Italy.

The Asian tiger mosquito, Aedes albopictus, has colonized nearly all the regions of Italy as well as other areas of Europe. During the summer of 2007 the tiger mosquito was responsible for an outbreak of Chikungunya in Italy, when this virus was brought in by a tourist of Indian origin returning from an endemic area. To increase the knowledge of tiger mosquito population dynamics, a survey was carried out from April to November 2008 in the municipalities of Arco and Riva del Garda (northern Italy) through a Biogents Sentinel™ (BG)-trap sampling. In particular, the aim of the study was to evaluate the influence of temperature and rainfall on the activity and dynamics of A. albopictus host-seeking females. The seasonal emergence of host-seeking females was strongly influenced by the minimum temperature, and a lower threshold of 13°C was identified. In addition, the threshold for the end of adult activity was found at a minimum temperature of 9°C. Host-seeking female abundance was positively affected by the accumulated temperatures over the period 3 and 4 weeks before the sampling week, possibly as a consequence of the positive effect of accumulated temperatures on larval density. Instead, accumulated precipitation over 1-4 weeks before sampling was negatively correlated with host-seeking female abundance. Finally, the activity of host-seeking females, estimated by the weekly increment in female abundance, was positively affected by the total abundance of females and by mean weekly temperatures. Our study provides useful information for predicting the dynamics of host-seeking Ae. albopictus females in northern Italy and for designing control strategies for preventing arbovirus outbreaks in areas colonized by Ae. albopictus.

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

Abstract. 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 12 390 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 I. 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.

Geographical Information Systems and Bootstrap Aggregation (Bagging) of Tree-Based Classifiers for Lyme Disease Risk Prediction in Trentino, Italian Alps

Abstract The risk of exposure to Lyme disease in the province of Trento, Italian Alps, was predicted through the analysis of the distribution of Ixodes ricinus (L.) nymphs infected with Borrelia burgdorferi s.l. with a model based on bootstrap aggregation (bagging) of tree-based classifiers within a geographical information system (GIS). Data on I. ricinus density assessed by dragging the vegetation in 438 sites during 1996 were cross-correlated with the digital cartography of a GIS, which included the variables altitude, exposure and slope, substratum, vegetation type and roe deer density. Ticks were more abundant at altitudes below 1,300 m a.s.l., in the presence of limestone and vegetation cover with thermophile deciduous forests and high densities of roe deer. A bootstrap aggregation procedure (bagging) was used to produce a model for the prediction of tick occurrence, the accuracy of which was tested on actual tick counts assessed by a further dragging campaign carried out during 1997 to determine infection prevalence and resulted in average 77%. Other tests of the model were made on additional and independent data sets. The prevalence of infection with Borrelia burgdorferi s.l, determined by polymerase chain reaction on 2,208 nymphs collected by random dragging in 245 transects selected within eight areas where the model predicted the occurrence of I. ricinus during 1997, was 17.5% and was positively correlated to tick abundance and roe deer density. These findings were used to relate the output of the bagged model (probability of tick occurrence) to the density of infected nymphs through a stepwise model selection procedure and thus to produce a GIS digital map of the probability distribution of infected nymphs in the Province of Trento at high resolution scale (50 by 50-m cell resolution). The application of the bagging procedure increased the accuracy of the prediction made by a single classification tree, a well-known classification method for the analysis of epidemiological data.