First report of the blood-feeding pattern in Aedes koreicus, a new invasive species in Europe.

Aedes koreicus is an invasive mosquito species which has been introduced into several European countries. Compared to other invasive Aedes mosquitoes, little is known of its biology and ecology. To determine Ae. koreicus' vectorial capacity, it is essential to establish its feeding patterns and level of anthropophagy. We report on the blood-feeding patterns of Ae. koreicus, examining the blood meal origin of engorged females and evaluating the influence of different biotic and abiotic factors on feeding behavior. Mosquitoes were collected in 23 sites in northern Italy by manual aspiration and BG-sentinel traps; host availability was estimated by survey. The source of blood meals was identified using a nested PCR and by targeting and sequencing the cytochrome c oxidase subunit I gene. In total, 352 Ae. koreicus engorged females were collected between 2013 and 2020 and host blood meals were determined from 299 blood-fed mosquitoes (84.9%). Eleven host species were identified, with the highest prevalences being observed among roe deer (Capreolus capreolus) (N = 189, 63.2%) and humans (N = 46, 15.4%). Blood meals were mostly taken from roe deer in forested sites and from humans in urban areas, suggesting that this species can feed on different hosts according to local abundance. Two blood meals were identified from avian hosts and one from lizard. Ae. koreicus' mammalophilic feeding pattern suggests that it may be a potential vector of pathogens establishing transmission cycles among mammals, whereas its role as a bridge vector between mammals and birds could be negligible.

A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020).

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Cytauxzoon sp. Infection and Coinfections in Three Domestic Cats in Central Italy.

Cytauxzoonosis is an emerging disease caused by a tick-transmitted haemoprotozoan affecting domestic and wild felids. The clinical and biomolecular findings of the infection due to Cytauxzoon sp. and concomitant coinfections are described in three cats in central Italy. Three domestic cats were referred for different clinical conditions (impact trauma, lameness, and weight loss and lethargy). They presented different hematobiochemical profiles. Only two cats were anemic, but in all three cats, endo erythrocyte inclusions suggestive of piroplasmids were found at blood smear evaluation. EDTA blood samples were submitted to rapid ELISA test for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), and to biomolecular investigations for Piroplasmida (Babesia spp., Theileria spp., Cytauxzoon spp.) and Mycoplasma spp. All three cats were positive for Cytauxzoon sp. (European Cytauxzoon species) and two cases were also coinfected by Candidatus Mycoplasma turicensis and FIV. This report suggests that cytauxzoonosis should be included among differential diagnoses in subjects with possibility of contact with ticks and with presence of coinfections by tick-borne parasites, including in non-endemic areas.

Clinical Tick-Borne Encephalitis in a Roe Deer (Capreolus capreolus L.).

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

Occurrence and Identification of Ixodes ricinus Borne Pathogens in Northeastern Italy.

In Europe, Ixodes ricinus is the main vector for tick-borne pathogens (TBPs), the most common tick species in Italy, particularly represented in pre-alpine and hilly northern areas. From 2011 to 2017, ticks were collected by dragging in Belluno province (northeast Italy) and analyzed by molecular techniques for TBP detection. Several species of Rickettsia spp. and Borrelia spp. Anaplaspa phagocitophilum, Neoerlichia mikurensis and Babesia venatorum, were found to be circulating in the study area carried by I. ricinus (n = 2668, all stages). Overall, 39.1% of screened pools were positive for at least one TBP, with a prevalence of 12.25% and 29.2% in immature stages and adults, respectively. Pathogens were detected in 85% of the monitored municipalities, moreover the presence of TBPs varied from one to seven different pathogens in the same year. The annual TBPs prevalence fluctuations observed in each municipality highlights the necessity of performing continuous tick surveillance. In conclusion, the observation of TBPs in ticks remains an efficient strategy for monitoring the circulation of tick-borne diseases (TBDs) in a specific area.

Mosquitoes of the Maculipennis complex in Northern Italy.

The correct identification of mosquito vectors is often hampered by the presence of morphologically indiscernible sibling species. The Maculipennis complex is one of these groups that include both malaria vectors of primary importance and species of low/negligible epidemiological relevance, of which distribution data in Italy are outdated. Our study was aimed at providing an updated distribution of Maculipennis complex in Northern Italy through the sampling and morphological/molecular identification of specimens from five regions. The most abundant species was Anopheles messeae (2032), followed by Anopheles maculipennis s.s. (418), Anopheles atroparvus (28) and Anopheles melanoon (13). Taking advantage of ITS2 barcoding, we were able to finely characterize tested mosquitoes, classifying all the Anopheles messeae specimens as Anopheles daciae, a taxon with debated rank to which we referred as species inquirenda (sp. inq.). The distribution of species was characterized by Ecological Niche Models (ENMs), fed by recorded points of presence. ENMs provided clues on the ecological preferences of the detected species, with An. daciae sp. inq. linked to stable breeding sites and An. maculipennis s.s. more associated to ephemeral breeding sites. We demonstrate that historical Anopheles malaria vectors are still present in Northern Italy.

Occurrence of Phlebotomine sand flies (Diptera: Psychodidae) in the northeastern plain of Italy.

BACKGROUND: Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. METHODS: The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO2-CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO2-CDC traps were identified by morphological and molecular analysis. RESULTS: From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus (n = 273), Sergentomyia minuta (n = 5), P. mascittii (n = 2) and P. perfiliewi (n = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from -2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. CONCLUSIONS: Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta, the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic.

The common European mosquitoes Culex pipiens and Aedes albopictus are unable to transmit SARS-CoV-2 after a natural-mimicking challenge with infected blood.

BACKGROUND: On 11 March 2020, the World Health Organisation (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak to be a pandemic. As the mosquito season progressed, the understandable concern that mosquitoes could transmit the virus began to increase among the general public and public health organisations. We have investigated the vector competence of Culex pipiens and Aedes albopictus, the two most common species of vector mosquitoes in Europe, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the very unusual feeding behaviour of Ae. albopictus, we also evaluated the role of this mosquito in a potential mechanical transmission of the virus. METHODS: For the vector competence study, mosquitoes were allowed to take several infectious blood meals. The mosquitoes were then collected and analysed at 0, 3, 7 and 10 days post-feeding. For the mechanical transmission test, Ae. albopictus females were allowed to feed for a short time on a feeder containing infectious blood and then on a feeder containing virus-free blood. Both mosquitoes and blood were tested for viral presence. RESULTS: Culex pipiens and Ae. albopictus were found not be competent vectors for SARS-CoV-2, and Ae. albopictus was unable to mechanically transmit the virus. CONCLUSIONS: This is the first study to show that the most common species of vector mosquitoes in Europe do not transmit SARS-CoV-2 and that Ae. albopictus is unable to mechanically transmit the virus from a positive host to a healthy host through host-feeding.

Echinococcus multilocularis and other cestodes in red foxes (Vulpes vulpes) of northeast Italy, 2012-2018.

BACKGROUND: Echinococcus multilocularis is a small tapeworm affecting wild and domestic carnivores and voles in a typical prey-predator life cycle. In Italy, there has been a focus of E. multilocularis since 1997 in the northern Italian Alps, later confirmed in red foxes collected from 2001 to 2005. In this study, we report the results of seven years of monitoring on E. multilocularis and other cestodes in foxes and describe the changes that occurred over time and among areas (eco-regions) showing different environmental and ecological features on a large scale. METHODS: Eggs of cestodes were isolated from feces of 2872 foxes with a sedimentation/filtration technique. The cestode species was determined through multiplex PCR, targeting and sequencing ND1 and 12S genes. Analyses were aimed to highlight variations among different eco-regions and trends in prevalence across the study years. RESULTS: Out of 2872 foxes, 217 (7.55%) samples resulted positive for cestode eggs at coproscopy, with differences of prevalence according to year, sampling area and age class. Eight species of cestodes were identified, with Taenia crassiceps (2.65%), Taenia polyacantha (1.98%) and E. multilocularis (1.04%) as the most represented. The other species, Mesocestoides litteratus, Taenia krabbei, T. serialis, T. taeniaeformis and Dipylidium caninum, accounted for < 1% altogether. Echinococcus multilocularis was identified in foxes from two out of six eco-regions, in 30 fecal samples, accounting for 1.04% within the cestode positives at coproscopy. All E. multilocularis isolates came from Bolzano province. Prevalence of cestodes, both collectively and for each of the three most represented species (T. crassiceps, T. polyacantha and E. multilocularis), varied based on the sampling year, and for E. multilocularis an apparent increasing trend across the last few years was evidenced. CONCLUSIONS: Our study confirms the presence of a focus of E. multilocularis in red foxes of northeast Italy. Although this focus seems still spatially limited, given its persistence and apparent increasing prevalence through the years, we recommend research to be conducted in the future on the ecological factors that, on a smaller scale, allow this zoonotic species to persist. On the same scale, we recommend a health education campaign to inform on the measures to prevent this zoonosis, targeted at people living in the area, especially hunters, dog owners, forestry workers and other potentially exposed categories.

Canine Leishmaniosis Control through the Promotion of Preventive Measures Appropriately Adopted by Citizens.

Canine leishmaniosis (CanL) is a disease caused by the protist Leishmania infantum and transmitted to dogs by sand fly (Diptera: Phlebotominae) bites. In 2005, a new autochthonous focus of CanL was recognised in the southern part of Euganei hills (northeastern Italy). In subsequent years, this outbreak was monitored, testing dogs and evaluating sand fly population. Moreover, dog owners were sensitized on the adoption of preventive measures, thanks to the collaboration of local administration, health authorities, and private veterinarians. This study includes serological tests on dogs, questionnaires submitted to dog owners regarding the use of preventive measures on their animals, and the evaluation of sand fly abundance. Data collected were statistically compared with those of previous years. The canine seroprevalence was significantly lower than that recorded at the beginning of the outbreak, despite the fact that sand fly abundance did not significantly decrease. In addition, most of the dog owners declared using regularly the topical insecticides on their dogs during the sand fly season. This experience demonstrated that a collaborative approach among scientific researchers, local authorities, and private veterinarians can achieve excellent results in the management of a leishmaniosis outbreak.

Survival of Rickettsia conorii in artificially contaminated whole and leukoreduced canine blood units during the storage period.

BACKGROUND: The ability of tick-borne agents to survive in stored blood bags is a key factor for their transmissibility by blood transfusion. The aim of this study was to evaluate the survival and potential infectivity of Rickettsia conorii (RC) in artificially contaminated canine whole blood (WB) and in leukoreduced whole blood (LR-WB) during the storage period. METHODS: RC was cultured on L929 cells. We used a one-week 25-cm2 flask with 70-80% of L929 infected cells to prepare the bacterial inoculum by pelleting cells and suspending the pellet in the donors' serum. We infected five 100 ml WB units with RC within 2 h from the collection and maintained it at room temperature for 4 h prior to refrigeration. We filtered 50 ml of each WB bag to obtain leukoreduced WB (LR-WB) at day 1 post-infection (dpi). We checked WB and LR-WB bags at 1, 4, 7, 14, 21, 28, 35 dpi for RC presence and viability through real-time PCR (rPCR) for DNA and mRNA, respectively, and by isolation. Identification of isolates was confirmed by indirect immunofluorescence and rPCRs. RESULTS: RC survived for the entire storage period in both whole and leukoreduced blood. All bags contained viable bacteria until 7 dpi; RC viability generally decreased over time, particularly in LR-WB bags where the isolation time was longer than in WB. Viable bacteria were still isolated at 35 dpi in 3 WB and 3 LR-WB. CONCLUSIONS: Leukoreduction reduced but did not eliminate RC in infected units. The survival and infectivity of RC in canine blood during the storage period may represent a threat for recipients.

A molecular survey of vector-borne pathogens and haemoplasmas in owned cats across Italy.

BACKGROUND: Feline vector-borne pathogens (FeVBPs) have been increasingly investigated for their impact on cat health and their zoonotic potential. The aim of the present study was to assess the prevalence of FeVBPs and haemoplasmas in cats across Italy and to identify potential risk factors linked to their occurrence. METHODS: Blood samples from 958 owned cats living in the North (n = 556), Centre (n = 173) and South (n = 229) of Italy were tested for Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp. and filarioids by conventional PCR (cPCR) and for haemoplasmas and Bartonella spp. by SYBR green real-time PCR. Cats included in the study represent a sub-sample from a larger number of animals enrolled in a previous study, which were selected based on the geographical origin. Data on cats' positivity for Leishmania infantum, feline leukaemia virus (FeLV) and for feline immunodeficiency virus (FIV), available from the previous study, were included and examined. Potential risk factors for pathogen infection were assessed in relationship to categorical variables including sex, geographical origin, breed, neutering status and age of cats. RESULTS: Out of the 958 cats, 194 (20.2%) were positive for at least one of the tested pathogens, 89 (16%) from the North, 32 (18.5%) from the Centre and 73 (31.9%) from the South of Italy. A high prevalence of FeVBPs was detected in male cats (n = 125, 27.8%), living in the southern part of the country (n = 73, 31.9%), younger than 18 months of age (n = 24, 22.4%) and not neutered (n = 39; 27.5%). In particular, 24 cats (2.5%) tested PCR-positive for Bartonella spp., of which 1.6% for B. henselae and 0.9% for B. clarridgeiae. A total of 111 cats scored PCR-positive for haemoplasmas (11.6%), specifically "Candidatus Mycoplasma haemominutum" (n = 95, 9.9%), M. haemofelis (n = 14, 1.5%) and "Candidatus Mycoplasma turicensis" (n = 2, 0.2%). Moreover, 39, 31 and 8 cats were positive for FeLV (4.1%), L. infantum (3.2%) and FIV (0.8%), respectively. Co-infections were registered for 19 (9.8%) cats. CONCLUSIONS: These results confirm the occurrence of haemoplasmas and FeVBPs throughout Italy. Preventive measures to protect both animal and human health should be carried out also for owned cats, even if no health status of animals has been assessed in this study.

Zoonotic and vector-borne pathogens in tigers from a wildlife safari park, Italy.

Infectious diseases by pathogens, including those of zoonotic concern, may act as a primary or contributory cause of threat to wildlife conservation and may represent a risk for human health, mainly for people working at, or visiting the zoological parks. Given the paucity of data on pathogens infecting wild tigers, we investigated the occurrence of infectious agents in this animal species, with a special focus on those of zoonotic concern. Blood and serum samples from tigers (n = 20) living in a wildlife safari park of southern Italy were screened by serological and molecular tests. All animals scored positive for antibodies against Toxoplasma gondii (100%), whereas they displayed different prevalence of seropositivity for Rickettsia conorii (30%), Bartonella henselae (15%) and Leptospira interrogans sv Icterohaemorrhagiae and/or Leptospira kirschneri sv Grippotyphosa (15%). No antibodies against Coxiella burnetii were detected. In addition, 8 tigers (40%) tested molecularly positive to "Candidatus Mycoplasma haemominutum", and 3 (15%) to Hepatozoon canis. No DNA of R. conorii, Bartonella spp., Ehrlichia/Anaplasma spp. and piroplasmids was amplified. The occurrence of tiger infections by bacteria and parasites may represent a risk for morbidity and, in some circumstances, mortality in this endangered species and a source of infection for other animals, including humans. These findings indicate that the circulation of zoonotic pathogens such as T. gondii, R. conorii, L. interrogans sv Icterohaemorrhagiae, "Candidatus Mycoplasma haemominutum" and B. henselae in given environments may represent a relevant health issue considering the close association among animals and humans visiting, or working at, the wildlife safari park. Preventative measures are advocated in order to control ectoparasites and other sources of infection (e.g., small rodents), thus for minimizing the risk of infection for animals as well as for humans.

Updates on Ecology and Life Cycle of Sulcascaris sulcata (Nematoda: Anisakidae) in Mediterranean Grounds: Molecular Identification of Larvae Infecting Edible Scallops.

Sulcascaris sulcata is a nematode parasite of sea turtles, widespread in neritic foraging grounds with variable prevalence, reaching 30% in loggerhead turtles Caretta caretta feeding in northern Adriatic Sea. Ulcerative gastritis associated to high intensity of infection is reported in this host species. The life cycle of S. sulcata has been elucidated in Australian and American waters, demonstrating the ability of the species of infecting a wide range of intermediate hosts, represented by bivalve and gastropod molluscs. During regular sanitary inspections, nematode larvae were found within the adductor muscle of Pecten jacobeus and Aequipecten opercularis collected from the Northern Adriatic Sea. Morphological and molecular analyses were performed for the identification of larvae, and molecular data were obtained from adult S. sulcata as well. Analysis of the sequences of ITS fragment, and mitochondrial genes cox1 and cox2, revealed high genetic similarity among all the samples, and no geographical clustering was observed between adult parasites collected in Adriatic and Tyrrhenian Seas. A common allele pool was detected also between the two developmental stages, included larvae from Pecten and Aequipecten. The comparison with other members of the family Anisakidae demonstrated that S. sulcata formed a clear monophyletic cluster. This study reports the first identification of intermediate hosts for S. sulcata within the Mediterranean Sea. Infection in edible scallops justifies the exclusion of the product from the market and zoonotic potential of larvae of this anisakid nematode are yet to be completely excluded. Fidelity of sea turtles to selected foraging grounds, such as the Northern Adriatic shelf, warrants the life cycle of S. sulcata to perpetrate in the area; at the same time, long distance migrations of individuals justify the dispersal of infecting elements over the Mediterranean basin, regardless of turtles' origin.

Cysticercosis by Taenia pisiformis in Brown Hare (Lepus europaeus) in Northern Italy: Epidemiologic and pathologic features.

In Northern Italy, a hastening of hare population decline was noticeable from 2008. In the same year hunters reported a sudden increase of hares infected by Taenia sp. larvae, whose morphology was consistent with T. pisiformis cysticerci. The aim of the survey was: i)to identify the parasites through morphological features and molecular techniques; ii)to quantify the prevalence and abundance of cysticerci in hunted hares; iii)to describe pathological aspects of parasite-induced lesions; iv)to evaluate the short-term trend of the infection comparing two different hunting seasons; v)to highlight possible relationship between T. pisiformis infection and hare-related variables. In 2013, 2015 the viscera of 54 and 61 hares legally hunted in agro-ecosystems of the Po Plain were collected. Peritoneum, liver and lungs were examined for cysticercosis; abundance was estimated counting superficial parasites in liver; parasites were microscopically identified by shape and measure of both large and small hooks. One cysticercus from each hare was analized by a PCR targeting Taeniid species and then sequenced. Frozen liver, lungs and gastrointestinal peritoneum were macroscopically observed and, after thawing, representative samples from the available organs were collected for histologic examination to verify parasitic cysts and the subsequent damage of the involved organs. Sex, weight and age class of the animals were recorded. Generalized linear models were used for statistical analysis. T. pisiformis was isolated in 8 hares in 2013 (prevalence 14.8%; abundance range: 0-400; mean abundance 17.8) and in 2 hares in 2015 (prevalence 3.28%; abundance range: 0-180; mean abundance 3.22). Identification was confirmed morphologically and by PCR. The DNA sequencing confirmed T. pisiformis in all samples. The sequences were all identical each-other. Infection was significantly related with adult age class, sampling year and low full-weight. Epidemiological and pathological pattern suggest both a possible role on host population health and a tendency toward host-parasite equilibrium.

A new approach to outbreak management for bovine Cystic Echinococcosis cases in hypo-endemic areas.

Cystic Echinococcosis (CE) surveillance in Italy is based on detection of its larval stage (hydatic cysts) at the slaughterhouse. In northern Italy, a hypo-endemic area, local health authorities investigate each individual farm with positive animals to treat their dogs with cestocidal drugs, but this system is time-consuming and poorly effective for bovine farms. The study applied a new approach based on targeted epidemiological surveys in areas with aggregation of bovine CE cases, and compared the outcome with that of two control areas with farms individually investigated. The presence of territorial cluster of bovine farms with CE cases was investigated for 3 consecutive years (2013-2014-2015) in a high-risk area of Veneto Region (north-eastern Italy), using a spatial scan statistic. Epidemiological investigations, consisting of a questionnaire survey and canine faecal samples collection, were conducted in cluster and control areas. All faecal samples were analyzed for taeniids eggs and positive samples were sequenced and identified. In total, 99 farms were surveyed and 208 faecal samples were retrieved from dogs. Sixty-two farms (42 bovine and 20 sheep) were investigated in cluster areas and 37 farms (33 bovine and 4 sheep) in control areas. Based on the results of the cestode egg isolation procedure, 14 animals (6.7%) were positive to taeniid eggs. For molecular analysis, two dogs resulted positive to Echinococcus granulosus, and seven to Taenia hydatigena. Twelve positive dogs were found in targeted survey areas and ten of these dogs were shepherd dogs, belonging to transhumant sheep flocks known to pass in cluster areas. The new approach demonstrated to successfully identify the probable source of infection of CE positive bovines. Most of positive dogs belonged to transhumant flocks, underlining the importance to include shepherd dogs in the surveillance system for CE.

Real-time PCR assay for screening Pneumocystis in free-living wild squirrels and river rats in Italy.

We used a real-time PCR (rtPCR) targeting a 150-bp amplicon of the mitochondrial small subunit of ribosomal RNA (mtSSU rRNA) to screen for Pneumocystis DNA in lungs of wild squirrels ( Callosciurus finlaysonii, n = 85) and river rats ( Myocastor coypus, n = 43) in Italy. The rtPCR revealed Pneumocystis DNA in 20 of 85 (24%) squirrels and in 35 of 43 (81%) river rats, and was more sensitive than a nested PCR that targets a portion of the mtSSU rRNA and the mitochondrial large subunit of rRNA (mtLSU rRNA). Phylogenetic analysis based on mtSSU rRNA and mtLSU rRNA sequences showed distinct Pneumocystis sequence types in these rodents. The rtPCR assay should be reliable for screening large populations for this potential pathogen, thereby allowing cost-effective monitoring of the disease in wild animals.

Exposure of Owned Dogs and Feeding Ticks to Spotted Fever Group Rickettsioses in Central Italy.

Dogs may be useful sentinels for public health monitoring of spotted fever group rickettsioses (SFGR). The aim of this study was to determine the exposure to SFGR among dogs and feeding ticks in central Italy. A total of 344 dogs and 607 adult ticks (395 Rhipicephalus sanguineus and 212 Ixodes ricinus specimens) collected from the coats of sampled animals were included in the study. Canine serum samples were analyzed by indirect fluorescent antibody technique (IFAT) for IgG antibodies against Rickettsia conorii and Rickettsia rickettsii. All the ticks and buffy coats were processed by a PCR targeting a fragment of gltA followed by sequencing. Overall, 56 dogs (16.3%) tested positive for one or both rickettsial antigens by IFAT with endpoint titers ranging from 1:64 to 1:2048; 38 (11%) serum samples reacted against R. conorii, 46 (13.4%) reacted against R. rickettsii, and 28 (8.1%) reacted simultaneously against both rickettsial agents. All buffy coats were PCR negative. Rickettsial DNA was revealed in 39 (18.4%) I. ricinus and in 10 (2.5%) R. sanguineus specimens. The amplicons sequencing showed three SFGR, that is, R. conorii detected in 10 R. sanguineus specimens and Rickettsia helvetica and Rickettsia monacensis detected in 7 and 32 I. ricinus ticks. Nine out of the 10 R. conorii isolates were obtained from ticks collected from seronegative dogs, and one specimen from a dog tested positive for both R. rickettsii and R. conorii by immunofluorescence assay. Among the seven ticks tested positive for R. helvetica, six were recovered from the coats of seronegative dogs and one from a dog having antibodies against R. conorii; the 32 isolates of R. monacensis were obtained from 28 seronegative and 4 R. conorii/R. rickettsii-positive dogs. The results highlight the non-negligible exposure of the canine population to SFGR in the sampled areas.

Ticks are more suitable than red foxes for monitoring zoonotic tick-borne pathogens in northeastern Italy.

BACKGROUND: Northeastern Italy is a hotspot for several tick-borne pathogens, transmitted to animals and humans mainly by Ixodes ricinus. Here we compare the results of molecular monitoring of ticks and zoonotic TBPs over a six-year period, with the monitoring of red foxes (Vulpes vulpes) in an endemic area. RESULTS: In the period 2011-2016, 2,578 ticks were collected in 38 sites of 20 municipalities of Belluno Province. Individual adults (264), pooled larvae (n = 330) and nymphs (n = 1984) were screened for tick-borne encephalitis virus, Borrelia burgdorferi (s.l.), Rickettsia spp., Babesia spp., Anaplasma phagocytophilum and "Candidatus Neoehrlichia mikurensis" by specific SYBR green real-time PCR assays and sequencing. The spleens of 97 foxes, culled in the period 2015-2017 during sport hunting or population control programs, were also screened. Overall, nine different pathogens were found in I. ricinus nymph and adult ticks: Rickettsia helvetica (3.69%); R. monacensis (0.49%); four species of the B. burgdorferi (s.l.) complex [B. afzelii (1.51%); B. burgdorferi (s.s.) (1.25%); B. garinii (0.18%); and B. valaisiana (0.18%)]; A. phagocytophilum (3.29%); "Candidatus N. mikurensis" (1.73%); and Babesia venatorum (0.04%). Larvae were collected and screened in the first year only and two pools (0.6%) were positive for R. helvetica. Tick-borne encephalitis virus was not found in ticks although human cases do occur in the area. The rate of infection in ticks varied widely according to tick developmental stage, site and year of collection. As expected, adults were the most infected, with 27.6% harboring at least one pathogen compared to 7.3% of nymphs. Pathogens with a minimum infection rate above 1% were recorded every year. None of the pathogens found in ticks were detectable in the foxes, 52 (54%) of which were instead positive for Babesia cf. microti (also referred to as Babesia microti-like, "Theileria annae", "Babesia annae" and "Babesia vulpes"). CONCLUSIONS: The results show that foxes cannot be used as sentinel animals to monitor tick-borne pathogens in the specific epidemiological context of northeastern Italy. The high prevalence of Babesia cf. microti in foxes and its absence in ticks strongly suggests that I. ricinus is not the vector of this pathogen.

First detection of Borrelia miyamotoi in Ixodes ricinus ticks from northern Italy.

BACKGROUND: Borrelia miyamotoi is a spirochete transmitted by several ixodid tick species. It causes a relapsing fever in humans and is currently considered as an emerging pathogen. In Europe, B. miyamotoi seems to occur at low prevalence in Ixodes ricinus ticks but has a wide distribution. Here we report the first detection of B. miyamotoi in Ixodes ricinus ticks collected in two independent studies conducted in 2016 in the north-eastern and north-western Alps, Italy. RESULTS: Three out of 405 nymphs (0.74%) tested positive for Borrelia miyamotoi. In particular, B. miyamotoi was found in 2/365 nymphs in the western and in 1/40 nymphs in the eastern alpine area. These are the first findings of B. miyamotoi in Italy. CONCLUSIONS: Exposure to B. miyamotoi and risk of human infection may occur through tick bites in northern Italy. Relapsing fever caused by Borrelia miyamotoi has not yet been reported in Italy, but misdiagnoses with tick-borne encephalitis, human granulocytic anaplasmosis or other relapsing fever can occur. Our findings suggest that B. miyamotoi should be considered in the differential diagnosis of febrile patients originating from Lyme borreliosis endemic regions. The distribution of this pathogen and its relevance to public health need further investigation.