Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

High-throughput screening of pathogens in Ixodes ricinus removed from hosts in Lombardy, northern Italy.

Ticks are important vectors of many pathogens in Europe, where the most impactful species is Ixodes ricinus. Recently, the geographical distribution of this tick species has been expanding, resulting in an increased risk of human exposure to tick bites. With the present study, we aimed to screen 350 I. ricinus specimens collected from humans and wild animals (mainly ungulates), to have a broader understanding of the tick-borne pathogens circulating in the Lombardy region, in northern Italy. To do so, we took advantage of a high-throughput real-time microfluidic PCR approach to screen ticks in a cost-effective and time-saving manner. Molecular analysis of the dataset revealed the presence of four genera of bacteria and two genera of protozoa: in ungulates, 77 % of collected ticks carried Anaplasma phagocytophilum, while the most common pathogen species in ticks removed from humans were those belonging to Borrelia burgdorferi sensu lato group (7.6 %). We also detected other pathogenic microorganisms, such as Rickettisa monacensis, Rickettsia helvetica, Neoehrlichia mikurensis, Babesia venatorum, and Hepatozoon martis. Besides, we also reported the presence of the pathogenic agent Borrelia miyamotoi in the area (1.4 % overall). The most common dual co-infection detected in the same tick individual involved A. phagocytophilum and Rickettsia spp. Our study provided evidence of the circulation of different tick-borne pathogens in a densely populated region in Italy.

The evolution of intramitochondriality in Midichloria bacteria.

Midichloria spp. are intracellular bacterial symbionts of ticks. Representatives of this genus colonise mitochondria in the cells of their hosts. To shed light on this unique interaction we evaluated the presence of an intramitochondrial localization for three Midichloria in the respective tick host species and generated eight high-quality draft genomes and one closed genome, showing that this trait is non-monophyletic, either due to losses or multiple acquisitions. Comparative genomics supports the first hypothesis, as the genomes of non-mitochondrial symbionts are reduced subsets of those capable of colonising the organelles. We detect genomic signatures of mitochondrial tropism, including the differential presence of type IV secretion system and flagellum, which could allow the secretion of unique effectors and/or direct interaction with mitochondria. Other genes, including adhesion molecules, proteins involved in actin polymerisation, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, to fuse with the organelles or manipulate the mitochondrial network.

Characterization of the bacterial microbiome of Swedish ticks through 16S rRNA amplicon sequencing of whole ticks and of individual tick organs.

BACKGROUND: The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. METHODS: In the present study the microbial flora of whole Ixodes ricinus, I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. RESULTS: The most abundant bacteria belonged to the group of Proteobacteria (Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus-associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. CONCLUSIONS: These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models.

Molecular Prevalence of Selected Tick-Borne Pathogens in Dermacentor reticulatus Collected in a Natural Park in Italy.

Dermacentor reticulatus is one of the most important vectors of tick-borne pathogens (TBPs) in Europe causing diseases in animals and humans. A longitudinal study was planned, aimed to detect the molecular prevalence of tick-borne pathogens, i.e., Babesia spp. and the spotted fever group Rickettsiae, and its seasonal variation in D. reticulatus questing ticks to define the temporal infection risk. Ticks were collected monthly over a period of 15 months in a peri-urban park in Lombardy, Italy. DNA extraction and molecular analyses were performed. Statistical analysis was carried out. Out of 488, 53 (P = 10.9%) adult questing ticks were positive for Babesia DNA. A higher prevalence was revealed in male (32/241, P = 13.3%) than in female (21/247, P = 8.5%) ticks. Positive ticks were mostly collected in winter months (P = 13.3%) compared to early (P = 7.9) and late (P = 12.8) spring months. A similar percentage of positive ticks was evidenced in transects 1 and 3 (5.8% and 6.5%, respectively); instead, a significant higher prevalence was recorded in transect 2 (P = 16.0%). Obtained sequences confirmed a homology of 100% with B. canis sequences deposited in GenBank. No ticks tested positive for Rickettsia spp. DNA (0/488, P = 0%). The conspicuous circulation of B. canis infection in D. reticulatus adult questing ticks confirms their role in the epidemiology of canine babesiosis and requires preventive measures for dogs in this recreational area. Even if no tick was positive for the spotted fever group Rickettsia, its capacity as a vector of zoonotic pathogens should not be neglected.

OCCURRENCE OF EUSTRONGYLIDES EXCISUS (NEMATODA:DIOCTOPHYMATIDAE) IN EUROPEAN PERCH (PERCA FLUVIATILIS) AND GREAT CORMORANT (PHALACROCORAX CARBO) IN LAKE ANNONE, NORTHERN ITALY.

The genus Eustrongylides includes zoonotic nematodes that infect fish species and fish-eating birds of freshwater ecosystems. This study aimed to evaluate the occurrence of Eustrongylides in the paratenic host Perca fluviatilis (European perch) and in the definitive host, Phalacrocorax carbo sinensis (great cormorant), in Lake Annone, a shallow eutrophic lake located in the pre-mountainous area of the Alps in northwest Italy where wintering cormorants coexist with new breeding colonies. A total of 114 European perch and 48 cormorants were examined for the occurrence of Eustrongylides. All parasites collected were identified with microscopic examination and molecular analysis. Overall, 11 specimens of European perch (9.6%) and 13 individuals of cormorants (27%) harbored nematodes identified as fourth-stage larvae and adults of Eustrongylides excisus. The observed prevalence of Eustrongylides spp. appears to be intermediate between the higher values in cormorant breeding areas in northern Europe and the lower prevalence observed in their wintering sites in southernmost Europe. Considering the eutrophication status of freshwater ecosystems and the increasing population of the cormorants, Eustrongylides has an increasing potential range of dispersion in Europe, including Italy; thus an extensive surveillance should be carried out, especially given the zoonotic potential of this nematode.

Molecular identification of Contracaecum rudolphii A and B (Nematoda: Anisakidae) from cormorants collected in a freshwater ecosystem of the pre-alpine area in Northern Italy.

Contracaecum rudolphii (s.l.) is a complex of sibling species with different genetic structure and ecological preference. This study reports the presence of specimens of Contracaecum rudolphii (s.l.) from sedentary and wintering cormorants (Phalacrocorax carbo sinensis) from the pre-mountain area of the Alps in Northern Italy, an important crossroads for most of the bird migration routes. A total of 48 specimens of cormorants collected from two adjacent freshwater habitats were analysed and C. rudolphii nematodes were retrieved in 100% of the examined specimens. A subsamples of 115 C. rudolphii individuals were genetically characterized and found to belong to the sibling species C. rudolphii B (n = 90) and C. rudolphii A (n = 25). C. rudolphii B were retrieved from both locations and included adults as well as larvae, while only adults of C. rudolphii A were detected, and in just one location. As expected for a freshwater environment, C. rudolphii B constitutes the largest sibling fraction, indicating that this likely is the endemic species, while cormorants originating from the breeding brackish lagoons and marine coastal environments of central and northern Europe could have brought C. rudolphii A from their breeding sites or migration stopovers.

A dual endosymbiosis supports nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Investigation of Tick-Borne Pathogens in Ixodes ricinus in a Peri-Urban Park in Lombardy (Italy) Reveals the Presence of Emerging Pathogens.

Ticks are important vectors of a great range of pathogens of medical and veterinary importance. Lately, the spread of known tick-borne pathogens has been expanding, and novel ones have been identified as (re)emerging health threats. Updating the current knowledge on tick-borne pathogens in areas where humans and animals can be easily exposed to ticks represents a starting point for epidemiological studies and public awareness. A PCR screening for tick-borne pathogens was carried out in Ixodes ricinus ticks collected in a peri-urban recreational park in Ticino Valley, Italy. The presence of Rickettsia spp., Borrelia burgdorferi senso latu complex, Anaplasma spp. and Babesia spp. was evaluated in a total of 415 I. ricinus specimens. Rickettsia spp. (R monacensis and R. helvetica) were detected in 22.96% of the samples, while B. burgdorferi s.l. complex (B. afzelii and B. lusitaniae) were present in 10.94%. Neoehrlichia mikurensis (1.99%) and Babesia venatorum (0.73%) were reported in the area of study for the first time. This study confirmed the presence of endemic tick-borne pathogens and highlighted the presence of emerging pathogens that should be monitored especially in relation to fragile patients, the difficult diagnosis of tick-borne associated diseases and possible interactions with other tick-borne pathogens.

Multi-country investigation of the diversity and associated microorganisms isolated from tick species from domestic animals, wildlife and vegetation in selected african countries.

In many areas of Africa, recent studies highlighted the great impact of ticks on animal and human health throughout the continent. On the other hand, very limited information on the bacterial endosymbionts of the African ticks and their pattern of co-infections with other bacteria are found in literature, notwithstanding their pivotal role in tick survival and vector efficiency. Thus, we investigated the distribution of selected pathogenic and symbiotic bacteria in hard ticks collected from wild, domestic animals and from vegetation in various ecological zones in Africa and their co-occurrence in the same tick host. Overall, 339 hard ticks were morphologically identified as belonging to the genera Amblyomma, Dermacentor, Hyalomma, Haemaphysalis, Ixodes and Rhipicephalus. Molecular screening provided information on pathogens circulation in Africa, detecting spotted fever group rickettsiae, Anaplasma spp., Ehrlichia ruminantium, Borrelia garinii, Babesia spp., Theileria spp. and Coxiella burnetii. Furthermore, our work provides insights on the African scenario of tick-symbiont associations, revealing the presence of Coxiella, Francisella and Midichloria across multiple tick populations. Coxiella endosymbionts were the most prevalent microorganisms, and that with the broadest spectrum of hosts, being detected in 16 tick species. Francisella was highly prevalent among the Hyalomma species tested and correlated negatively with the presence of Coxiella, showing a potential competitive interaction. Interestingly, we detected a positive association of Francisella with Rickettsia in specimens of Hy. rufipes, suggesting a synergistic interaction between them. Finally, Midichloria was the most prevalent symbiont in Rhipicephalus sanguineus sensu lato from Egypt.

First detection of Amblyomma variegatum and molecular finding of Rickettsia africae in Sardinia, Italy.

Here we present the first detection of a male Amblyomma variegatum tick infesting a sheep on the island of Sardinia, as well as the detection of a pathogen, Rickettsia africae, in DNA extracted from this tick. The tick, the second individual of this species reported in Italy (the first one was reported in Sicily by Albanese in 1971) was collected in August 2018 from the inguinal region of an adult female sheep in a farm located near Sassari (North-West Sardinia). The tick was identified as an adult A. variegatum male under a stereomicroscope using morphological keys. A phylogenetic analysis showed that the 12S sequence clustered with that of African A. variegatum individuals and was embedded within the previously identified West African group. We tested the tick for the presence of microorganisms of the genera Ehrlichia, Rickettsia, Anaplasma, Theileria and Babesia, using published PCR protocols. The tick was found positive to Rickettsia and the obtained sequence matched at 100 % identity with R. africae. The area where the tick was detected was inspected on multiple occasions, looking for other specimens of A. variegatum, without any results. In the same period another male specimen of A. variegatum was found in Haute Corse in 2019. The authors' hypothesis is that the presence of the A. variegatum specimen is an occasional finding, probably linked to the migrating birds that cross Sardinia and Corsica from Africa during summer. Although this may have been an incidental finding, it must be considered that global warming could increase the risk of establishment of colonies of these ticks, that show a strong spreading capability. It is also important to emphasize that this tick species is a proven vector and reservoir of R. africae, an uncommon zoonotic pathogen in Italy, thus additional monitoring must be performed as the establishment of a stable population in Sardinia could represent a serious veterinary and public health issue.

Sequence of a Coxiella Endosymbiont of the Tick Amblyomma nuttalli Suggests a Pattern of Convergent Genome Reduction in the Coxiella Genus.

Ticks require bacterial symbionts for the provision of necessary compounds that are absent in their hematophagous diet. Such symbionts are frequently vertically transmitted and, most commonly, belong to the Coxiella genus, which also includes the human pathogen Coxiella burnetii. This genus can be divided in four main clades, presenting partial but incomplete cocladogenesis with the tick hosts. Here, we report the genome sequence of a novel Coxiella, endosymbiont of the African tick Amblyomma nuttalli, and the ensuing comparative analyses. Its size (∼1 Mb) is intermediate between symbionts of Rhipicephalus species and other Amblyomma species. Phylogenetic analyses show that the novel sequence is the first genome of the B clade, the only one for which no genomes were previously available. Accordingly, it allows to draw an enhanced scenario of the evolution of the genus, one of parallel genome reduction of different endosymbiont lineages, which are now at different stages of reduction from a more versatile ancestor. Gene content comparison allows to infer that the ancestor could be reminiscent of C. burnetii. Interestingly, the convergent loss of mismatch repair could have been a major driver of such reductive evolution. Predicted metabolic profiles are rather homogenous among Coxiella endosymbionts, in particular vitamin biosynthesis, consistently with a host-supportive role. Concurrently, similarities among Coxiella endosymbionts according to host genus and despite phylogenetic unrelatedness hint at possible host-dependent effects.

Where to find questing Ixodes frontalis ticks? Under bamboo bushes!

Tick-borne diseases have a complex epidemiology that depends on different ecological communities, associating several species of vertebrate hosts, vectors and pathogens. While most studies in Europe are focused on Ixodes ricinus, other Ixodes species may also be involved in the transmission or maintenance of pathogens. This is the case of Ixodes frontalis, a poorly known species associated with different bird species such as blackbirds, thrushes and robins, with a wide distribution covering most European countries. In a previous study, high densities of questing I. frontalis larvae were found during autumn-winter at a site close to Nantes (western France) where a long-term survey focused on I. ricinus was conducted. These I. frontalis were mostly observed under bamboo bushes. In the present study, we investigated the presence of I. frontalis under bamboo bushes at various locations. With that aim in mind, a systematic search for questing I. frontalis was undertaken by the flagging method in public urban parks and private gardens presenting bamboo bushes (32 sites). This survey was carried out during autumn-winter to maximize the probability of finding the most abundant stage, i.e. larvae. We searched for I. frontalis first in the area of Nantes (10 sites), then in other regions of France (21 sites) and at one site in northern Italy. A single visit to each site revealed the presence of I. frontalis at 29 out of 32 sites: larvae were always present, nymphs were frequent (59 % of the positive sites), while adults were found at only 14 % of the sites. Questing stages of this understudied species are thus easy to find, by dragging or flagging under bamboo bushes in autumn or winter. We make the assumption that bamboo offers a favourable place for birds to roost overnight outside their breeding period (i.e. spring), sheltered from both predators and wind. This would explain higher densities of I. frontalis under bamboo, relative to other biotopes. As I. frontalis is known to harbour zoonotic pathogens, the consequences of this discovery on the epidemiology of tick-borne diseases are discussed.

Development of a PCR for Borrelia burgdorferi sensu lato, targeted on the groEL gene.

Borrelia burgdorferi sensu lato (s.l.) is the etiological agent of Lyme disease, transmitted by ticks of the genus Ixodes Latreille. Diagnosis of Lyme disease in humans is often difficult and a detailed knowledge of the circulation of B. burgdorferi s.l. in tick hosts is therefore fundamental to support clinical procedures. Here we developed a molecular approach for the detection of B. burgdorferi s.l. in North Italian Ixodes ricinus (Linnaeus). The method is based on the amplification of a fragment of the groEL gene, which encodes a heat-shock protein highly conserved among B. burgdorferi s.l. species. The tool was applied in both qualitative and Real-time PCR approaches testing ticks collected in a North Italian area. The obtained results suggest that this new molecular tool could represent a sensitive and specific method for epidemiological studies aimed at defining the distribution of B. burgdorferi s.l. in I. ricinus and, consequently, the exposure risk for humans.

Lyme borreliosis incidence in Lombardy, Italy (2000-2015): Spatiotemporal analysis and environmental risk factors.

Lyme borreliosis cases have been reported from Lombardy in northern Italy, where Ixodes ricinus is the main vector of Borrelia burgdorferi sensu lato. However, spatial and temporal variation in the incidence of Lyme borreliosis is not well understood. In the present study, based on new notified cases of Lyme borreliosis from 2000 to 2015, an average of 1.24 new cases per million residents per year was documented. New cases, georeferenced at the municipal level, were analyzed by retrospective space-time analysis (using SaTScan v. 9.3.1); and land cover, extrapolated from a Corine Land Cover dataset (using QGIS 2.8.1), was used to implement an environmental risk factor analysis. Firstly, a temporal high-risk cluster was detected in Lombardy: the relative risk of Lyme borreliosis was 3.73 times higher during 2008-2015 compared with the entire study period. Moreover, in a spatiotemporal high-risk cluster with a circular base, land cover consisting of wildland-urban interface, meadow, forest and meadow-forest transition were significantly higher compared to low-risk areas. Results of the present study demonstrate that the incidence of Lyme borreliosis is increasing in Lombardy and that environmental conditions are suitable for I. ricinus ticks infected with B. burgdorferi s.l.: citizens and health systems should be aware of Lyme borreliosis to reduce tick bites with personal protective behaviors and to avoid misdiagnosis, particularly within the area including the observed high-risk cluster. Economic resources should be invested to inform about methods to prevent tick bites, how to check people and pets after frequenting risk areas, and ways of removing the biting ticks when they are found.

Tissue tropism and metabolic pathways of Midichloria mitochondrii suggest tissue-specific functions in the symbiosis with Ixodes ricinus.

A wide range of arthropod species harbour bacterial endosymbionts in various tissues, many of them playing important roles in the fitness and biology of their hosts. In several cases, many different symbionts have been reported to coexist simultaneously within the same host and synergistic or antagonistic interactions can occur between them. While the associations with endosymbiotic bacteria have been widely studied in many insect species, in ticks such interactions are less investigated. The females and immatures of Ixodes ricinus (Ixodidae), the most common hard tick in Europe, harbour the intracellular endosymbiont "Candidatus Midichloria mitochondrii" with a prevalence up to 100%, suggesting a mutualistic relationship. Considering that the tissue distribution of a symbiont might be indicative of its functional role in the physiology of the host, we investigated M. mitochondrii specific localization pattern and the corresponding abundance in selected organs of I. ricinus females. We paired these experiments with in silico analysis of the metabolic pathways of M. mitochondrii, inferred from the available genome sequence, and additionally compared the presence of these pathways in seven other symbionts commonly harboured by ticks to try to obtain a comparative understanding of their biological effects on the tick hosts. M. mitochondrii was found to be abundant in ovaries and tracheae of unfed I. ricinus, and in ovaries, Malpighian tubules and salivary glands of semi-engorged females. These results, together with the in silico metabolic reconstruction allow to hypothesize that the bacterium could play multiple tissue-specific roles in the host, both enhancing the host fitness (supplying essential nutrients, enhancing the reproductive fitness, helping in the anti-oxidative defence, in the energy production and in the maintenance of homeostasis and water balance) and/or for ensuring its presence in the host population (nutrients acquisition, vertical and horizontal transmission). The ability of M. mitochondrii to colonize different tissues allows to speculate that distinctive sub-populations may display different specializations in accordance with tissue tropism. Our hypotheses should be corroborated with future nutritional and physiological experiments for a better understanding of the mechanisms underlying this symbiotic interaction.

Gastrointestinal Nematodes of Goats: Host-parasite Relationship Differences in Breeds at Summer Mountain Pasture in Northern Italy.

INTRODUCTION: The Orobic goat is a hardy breed native to the Orobic Alps (Lombardy, northern Italy). The aim of the study was the assessment of gastrointestinal nematode (GIN) egg excretion in Alpine and Saanen (cosmopolite breeds) and Orobic grazing goats, after a strategic treatment with eprinomectin in late June. MATERIAL AND METHODS: Individual faecal samples from a mixed flock of cosmopolite and Orobic goats were collected and analysed by the FLOTAC double technique every three weeks from June to September. RESULTS: Strongylida was the primary GIN infection observed in goats that grazed on Alpine pastures; a strategic treatment with eprinomectin led to a prolonged reduction of egg excretion during the whole study period. Egg excretion was also influenced by breed. Pluriparous Orobic does were able to control reinfection better than the pluriparous cosmopolite does. Regarding Nematodirus sp. eggs per gram of faeces (EPG), the autochthonous Orobic breed presented higher values than the cosmopolite breeds. However, cosmopolite goats presented higher EPG values of Strongyloides papillosus than their Orobic counterparts in August. CONCLUSIONS: Further studies on genetic features of local autochthonous goats, such as the Orobic breed, are needed, since they could reveal peculiar characteristics of susceptibility, resistance or resilience to GIN infection, providing genetic resources for selection.

Toxoplasma gondii infection in raptors from Italy: Seroepidemiology and risk factors analysis.

Toxoplasma gondii is a zoonotic parasite infecting a wide range of intermediate hosts, including birds. Nevertheless, scant information on the spread of infection in wild bird populations is available to date. With the aim of updating information on T. gondii infection in birds of prey and possible risk factors associated with the infection, a serosurvey was planned on both wild and captive raptors. An overall of 93 raptors from Northern Italy were tested for the presence of anti-T. gondii antibodies with a commercial modified agglutination test (MAT). A T. gondii prevalence of 10.7% was recorded; the highest seroprevalence was observed within the Family Strigidae (12.5%). Only wild animals tested positive; any statistical difference among species, taxonomic family, age, origin, use, migratory behavior and diet composition was not highlighted. Toxoplasmosis in birds of prey, sentinel species for the environmental spread of T. gondii, should always be considered within sanitary programs devoted to avian species protection.

Transmission of Rickettsia raoultii and Rickettsia massiliae DNA by Dermacentor reticulatus and Rhipicephalus sanguineus (s.l.) ticks during artificial feeding.

BACKGROUND: Tick-borne rickettsial pathogens are emerging worldwide and pose an increased health risk to both humans and animals. A plethora of rickettsial species has been identified in ticks recovered from human and animal patients. However, the detection of rickettsial DNA in ticks does not necessarily mean that these ticks can act as vectors for these pathogens. Here, we used artificial feeding of ticks to confirm transmission of Rickettsia massiliae and Rickettsia raoultii by Rhipicephalus sanguineus (sensu lato) and Dermacentor reticulatus ticks, respectively. The speed of transmission was also determined. METHODS: An artificial feeding system based on silicone membranes were used to feed adult R. sanguineus (s.l.) and D. reticulatus ticks. Blood samples from in vitro feeding units were analysed for the presence of rickettsial DNA using PCR and reverse line blot hybridisation. RESULTS: The attachment rate of R. sanguineus (s.l.) ticks were 40.4% at 8 h post-application, increasing to 70.2% at 72 h. Rickettsia massiliae was detected in blood samples collected 8 h after the R. sanguineus (s.l.) ticks were placed into the in vitro feeding units. D. reticulatus ticks were pre-fed on sheep and subsequently transferred to the in vitro feeding system. The attachment rate was 29.1 % at 24 h post-application, increasing to 43.6 % at 96 h. Rickettsia raoultii was detected in blood collected 24 h after D. reticulatus was placed into the feeding units. CONCLUSIONS: Rhipicephalus sanguineus (s.l.) and D. reticulatus ticks are vectors of R. massiliae and R. raoultii, respectively. The transmission of R. massiliae as early as 8 h after tick attachment emphasises the importance of removing ticks as soon as possible to minimise transmission. This study highlights the relevance of in vitro feeding systems to provide insight into the vectorial capacity of ticks and the dynamics of tick-borne pathogen transmission.

Arthropods and associated pathogens from native and introduced rodents in Northeastern Italy.

Introduced Siberian chipmunks Eutamias sibiricus have been reported to be important reservoirs for human Lyme disease, as they may host high numbers of hard ticks carrying Borrelia spp. and other pathogens. In the present study, we assessed the prevalence of Borrelia spp. and other pathogenic bacteria in ectoparasite arthropod species infesting Siberian chipmunks and coexisting native small rodents. Small rodents were trapped with Sherman traps in Veneto (NE Italy), where the largest Italian populations of chipmunks occur. A total of 14 individual ticks were found on 223 rodents, with 6 more ticks obtained from stored dead chipmunks from the same study area. Ectoparasites were screened for pathogens by molecular analyses including species-specific PCR amplifications. Rickettsia monacensis, Borrelia lusitaniae, and Anaplasma platys were present in the parasites of both native rodents and introduced chipmunks. The present findings suggest a role for the invasive species E. sibiricus in the maintenance of the Ixodes ricinus life cycle, which may result in the modification of the transmission dynamics of tick-borne pathogens. Moreover, the presence of Rickettsia in urban populations of chipmunks may represent a serious risk for human health and should be investigated further.