An early start of West Nile virus seasonal transmission: the added value of One Heath surveillance in detecting early circulation and triggering timely response in Italy, June to July 2018.

In Italy, the 2018 West Nile virus transmission season started early with a high number of cases reported. One-Health surveillance, within the Italian West Nile national preparedness and response plan, detected viral circulation 9 days before symptom-onset of the first confirmed human case; triggering timely implementation of blood and transplant safety measures. This is an example of how functional coordination allows health authorities to use early warning triggers from surveillance systems to implement preventive measures.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF AMBLYOMMA SCUTATUM (ACARI: IXODIDAE) ACCIDENTALLY INTRODUCED IN ITALY.

Eight ticks were found in Comacchio (FE), Italy parasitizing a young black iguana (Ctenosaura similis) that had been accidentally transported in a commercial plant container from Costa Rica. Specimens were identified morphologically as Amblyomma scutatum and then confirmed by the barcoding of the mitochondrial cytochrome c oxidase subunit 1 gene. Amblyomma scutatum is a common tick known to infest reptiles in Central America, Mexico, and Venezuela, but not in Europe. In Italy, the possibility for this tick to become endemic is unlikely because of the absence of its principal hosts. Nevertheless, this finding confirms the high risk of introducing exotic species that is linked with global commerce and therefore the need for veterinary control of shipments.

In vitro susceptibility of Brachyspira hyodysenteriae strains isolated in pigs in northern Italy between 2005 and 2022.

Pleuromutilins (tiamulin and valnemulin) are often used to treat swine dysentery due to recurrent resistance to macrolides and lincosamides. Recently, reduced susceptibility of B. hyodysenteriae to pleuromutilin has been reported. 536 strains of B. hyodysenteriae were isolated from symptomatic pigs weighing 30-150 kg in northern Italy between 2005 and 2022. B. hyodysenteriae was isolated by standard methods and confirmed by PCR. The minimum inhibitory concentration (MIC) to doxycycline, lincomycin, tiamulin, tylosin, tylvalosine and valnemulin was evaluated according to CLSI procedures and MIC data were reported as MIC 50 and MIC 90. The temporal trend of the MIC values was evaluated by dividing the data into two groups (2005-2013 and 2014-2022). Comparison of the distribution in frequency classes in the two periods was performed using Pearson's chi-squared test (p < 0.01). MIC 50 was close to the highest values tested for lincomycin and tylosin, while MIC 90 was close to the highest values tested for all antibiotics. 71.7% of the strains were susceptible to tylvalosin, while 75%-80.4% had reduced susceptibility to valnemulin and tiamulin, respectively. The difference in the distribution of MIC classes was statistically significant in the two periods for doxycycline, tiamulin, tylvalosin and valnemulin, and more MIC classes above the epidemiological cut-off were observed in 2014-2022 compared with 2005-2013. The evaluation of the trends during the period considered shows a decreasing rate of wild-type strains with MIC values below the epidemiological cut-off over time and confirms the presence of resistant strains in northern Italy.

Arbovirus screening of mosquitoes collected in 2022 in Emilia-Romagna, Italy, with the implementation of a real-time PCR for the detection of Tahyna virus.

Several Arboviruses (Arthropod-borne virus) are a concrete health risk. While some arboviruses, such as the West Nile virus (WNV) and the Usutu virus (USUV) are actively surveyed, others are neglected, including the Tahyna virus (TAHV). In this work, we tested - searching for all the three viruses - 37,995 mosquitoes collected in 95 attractive traps, baited by carbon dioxide, distributed in the lowlands of Emilia-Romagna, northern Italy, between 19 July and 12 August 2022. Among the 668 pools obtained, WNV was detected in 45 pools of Culex (Cx.) pipiens and USUV was recorded in 24 pools of the same mosquito; ten of these Cx. pipiens pools tested positive for both WNV and USUV. Interestingly, we recorded a significant circulation of both WNV lineage 1 (WNV-L1) and lineage 2 (WNV-L2): WNV-L1 strains were detected in 40 pools, WNV-L2 strains in three pools and both lineages were detected in two pools. TAHV was detected in 8 different species of mosquitoes in a total of 37 pools: Aedes (Ae.) caspius (25), Ae. albopictus (5), Ae. vexans (3), Cx. pipiens (2), Ae. cinereus (1) and Anopheles maculipennis sl (1). The significant number of Ae. caspius-pools tested positive and the estimated viral load suggest that this mosquito is the principal vector in the surveyed area. The potential involvement of other mosquito species in the TAHV cycle could usefully be the subject of further experimental investigation. The results obtained demonstrate that, with adequate sampling effort, entomological surveillance is able to detect arboviruses circulating in a given area. Further efforts must be made to better characterise the TAHV cycle in the surveyed area and to define health risk linked to this virus.

Monitoring of Insecticide Resistance Mutations and Pathogen Circulation in Sand Flies from Emilia-Romagna, a Leishmaniasis Endemic Region of Northern Italy.

The continuously expanding distribution of sand flies, proven vectors of Leishmania and of several phleboviruses, is a growing public health issue in Europe. Especially in Italy, visceral leishmaniasis (VL) is occurring with increasing incidence northward, in previously non-endemic provinces. Around the globe, disease elimination efforts largely focus on sand fly vector insecticidal control, often leading to the development of resistance. In Emilia-Romagna (ER), northern Italy, insecticides are heavily applied for agricultural and mosquito control, but not specifically against sand flies. Here, we investigated the sand fly species composition in certain environmental settings in ER provinces and monitored the presence of pyrethroid resistance mutations and pathogen circulation. Phlebotomus perfiliewi, a dominant vector of Leishmania infantum, was detected almost exclusively in the region. No mutations in the voltage-gated sodium channel gene, e.g., knock-down resistance mutations I1011M, L1014F/S, V1016G, or F1020S, were recorded. Pathogen monitoring revealed that almost 40% of the tested sand fly pools were positive for Leishmania, while the presence of Toscana and Fermo phleboviruses was also observed in much lower frequencies (≤3% positive pools). Regular epidemiological and entomological monitoring, alongside resistance surveillance, is highly recommended to ensure the sustainability and efficiency of vector control interventions.

Effects of the Weather on the Seasonal Population Trend of Aedes albopictus (Diptera: Culicidae) in Northern Italy.

BACKGROUND: Aedes albopictus, the Asian tiger mosquito, has become a prevalent pest in Italy, causing severe nuisance and posing a threat of transmission of arboviruses introduced by infected travelers. In this study, we investigated the influence of weather parameters on the seasonal population density of Aedes albopictus. METHODS: A Bayesian approach was employed to identify the best meteorological predictors of species trend, using the eggs collected monthly from 2010 to 2022 by the Emilia-Romagna regional monitoring network. RESULTS: The findings show that the winter-spring period (January to May) plays a crucial role in the size of the first generation and seasonal development of the species. CONCLUSIONS: A temperate winter and a dry and cold March, followed by a rainy and hot spring and a rainy July, seem to favor the seasonal development of Ae. albopictus.

New Isolation of Ponticelli III Virus (Bunyavirales: Phenuiviridae) in Emilia-Romagna Region, Italy.

The number of newly described sandfly-borne phleboviruses has been steadily growing in recent years. Some phleboviruses are human pathogens, but their health relevance is largely uncharacterized. We aimed to investigate the circulation of these viruses in the Emilia-Romagna region where several have already been described. A total of 482 sandflies were collected in a site in Reggio Emilia in 2019 and 2020. Sandflies collected in 2020 were grouped in 21 pools with a maximum of 25 sandflies per pool, submitted to real time PCR, and isolated in Vero cell culture. Complete genome sequencing showed the isolation of a strain of a Ponticelli III virus. This virus, which belongs to the species Adana phlebovirus, differed in the M segment from the Ponticelli I and Ponticelli II viruses. Analysis performed on the genomic segments of the newly isolated virus compared with other phleboviruses highlighted a strong purifying selection in the L segments, and different substitution saturation, highest in the M segments. Future research should address the ecological processes driving the occurrence of these novel phleboviruses and their possible impact on public health.

Identification and phylogenetic analysis of Taenia spp. parasites found in wildlife in the Emilia-Romagna region, northern Italy (2017-2022).

The recent expansion of the habitat of several wildlife species, comprising anthropized areas, is a relevant risk factor for many zoonotic diseases and should be considered in national and regional sanitary monitoring systems. We evaluated adult intestinal Taenia spp. parasites isolated from wild carnivores and cystic larval forms isolated from wild mammals analysed at the Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna (IZSLER) as part of the regional wildlife sanitary surveillance plan. Then, we assessed parasite species through molecular analysis (multiplex PCR followed by ribosomal 12S subunit gene sequencing) in order to update the epidemiological situation on Taeniids infection in the Emilia-Romagna wildlife, reporting the prevalence of each isolated species. The most commonly isolated species was Taenia serialis, which we detected in both wolves and foxes as definitive hosts and in roe deer as intermediate host. More attention on the distribution of Taeniids in wildlife should be paid, considering their potential zoonotic role: several Taenia spp. (Taenia solium, Taenia multiceps, Taenia serialis, Taenia brauni, Taenia glomerulatus) are known for causing coenurosis in humans, with possible severe or fatal outcomes.

Detection of mosquito-only flaviviruses in Europe.

The genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy, Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.

Etiological [corrected] agents of rickettsiosis and anaplasmosis in ticks collected in Emilia-Romagna region (Italy) during 2008 and 2009.

Ticks are the main vectors of rickettsiae of the spotted fever group, as well as of a variety of other Rickettsiales, including bacteria of the genus Anaplasma, that might cause diseases in humans and animals. Here we present the result of a survey for ticks and for tick-associated Rickettsiales in the Emilia Romagna region (Northern Italy). The study was focused on ticks collected from wild-hunted animals. Out of 392 ticks collected from these animals, 282 (72%) were identified as Ixodes ricinus, 110 (28%) as Dermacentor marginatus. The former was found on four vertebrate species, whereas the latter appeared more specific for wild boar. The presence of rickettsiae was demonstrated in 22.5% of I. ricinus (57/253) and in 29% of D. marginatus (32/110). Five ticks of the species I. ricinus were also positive for Anaplasma phagocytophilum (2%). In addition, we collected ticks by dragging in a natural park of the same region. All of the ticks captured by dragging were identified as I. ricinus. Thirty-six out of 200 analyzed ticks proved positive for Rickettsia monacensis and R. helvetica (16.5 and 1.5%, respectively). Our results highlight that that ticks present in wild areas, widely exploited for recreation and hunting in Emilia-Romagna, represent a risk for the transmission of spotted fevers and anaplasmosis to humans.

Host preference and Leishmania infantum natural infection of the sand fly Phlebotomus perfiliewi in northern Italy.

The host preference of hematophagous insects is important in determining the cycle of pathogens that they potentially transmit; for example, sand flies are competent vectors of Leishmania parasites. In this work, we evaluated the host preference of sand flies collected in the Emilia-Romagna region of Italy in 2018 and 2019 in an area in which Leishmania infantum circulates actively. Out of about 30,000 sampled sand flies, we obtained 252 engorged females, which were processed to identify the sources of blood meals. Sampling data collected confirmed a positive phototropism of Phlebotomus (Ph.) perfiliewi respect to Ph. perniciosus and the enhanced efficiency of light traps in collecting engorged females compared with traps baited with carbon dioxide. We identified blood source in 185 females (183 Ph. perfiliewi, two Ph. pernicious). The most bitten animal was the roe deer (49.5%), followed by humans (29.2%), hare (7.1%) and cow (4.7%). Other animals, including wild boar, horse, donkey, porcupine, chicken and red fox, were less represented (<2%), while the blood of dogs and rodents were not detected. In addition, we singly screened engorged females for Leishmania founding 5 positive specimens, fed on roe deer (4) and man (1), providing evidence of parasite circulation in a sylvatic environment, where presence of dogs was not common. These findings suggest the existence of an uncharacterized Leishmania reservoir in the surveyed area.

Fate of Salmonella enterica Typhimurium and Listeria monocytogenes in Black Soldier Fly (Hermetia illucens) Larvae Reared on Two Artificial Diets.

Ensuring food security is one of the main challenges facing the world over the next 30 years. There is, thus, an urgent need to significantly increase the supply of sustainable protein that can be transformed into animal feed. Proteins from insects offer a valuable alternative. This article presents the results of challenge tests conducted to investigate the dynamics of the microbial load of Salmonella enterica Typhimurium and Listeria monocytogenes in black soldier fly (Hermetia illucens) larvae grown on contaminated substrates. Four separate challenge tests were performed on two substrates: the Gainesville diet and a homemade diet. The challenge test procedure was carried out in accordance with ISO/DIS 20976-2 (under development). The results of this study show that, when grown on contaminated substrates, BSF larvae do not eliminate Salmonella Typhimurium or L. monocytogenes, but can reduce their microbial load. Sanitation processes downstream of the breeding of BSF larvae are, however, required to reduce the microbiological risks of this novel food.

Unsupervised machine learning and geometric morphometrics as tools for the identification of inter and intraspecific variations in the Anopheles Maculipennis complex.

Geometric morphometric analysis was combined with two different unsupervised machine learning algorithms, UMAP and HDBSCAN, to visualize morphological differences in wing shape among and within four Anopheles sibling species (An. atroparvus, An. melanoon, An. maculipennis s.s. and An. daciae sp. inq.) of the Maculipennis complex in Northern Italy. Specifically, we evaluated: (1) wing shape variation among and within species; (2) the consistencies between groups of An. maculipennis s.s. and An. daciae sp. inq. identified based on COI sequences and wing shape variability; and (3) the spatial and temporal distribution of different morphotypes. UMAP detected at least 13 main patterns of variation in wing shape among the four analyzed species and mapped intraspecific morphological variations. The relationship between the most abundant COI haplotypes of An. daciae sp. inq. and shape ordination/variation was not significant. However, morphological variation within haplotypes was reported. HDBSCAN also recognized different clusters of morphotypes within An. daciae sp. inq. (12) and An. maculipennis s.s. (4). All morphotypes shared a similar pattern of variation in the subcostal vein, in the anal vein and in the radio-medial cross-vein of the wing. On the contrary, the marginal part of the wings remained unchanged in all clusters of both species. Any spatial-temporal significant difference was observed in the frequency of the identified morphotypes.  Our study demonstrated that machine learning algorithms are a useful tool combined with geometric morphometrics and suggest to deepen the analysis of inter and intra specific shape variability to evaluate evolutionary constrains related to wing functionality.

Identification of Toscana virus in natural population of sand flies (Diptera: Psychodidae) from Moroccan leishmaniasis foci.

BACKGROUND: Phlebotomine sand flies are known as vectors of various pathogens such as Leishmania sp parasite and Toscana virus (TOSV). Leishmaniasis is endemic in Morocco, and TOSV is increasingly reported. Our objective is to analyze the specific composition of the natural population of sand flies in endemic and non endemic area of leishmaniasis in Morocco, thus evaluated their infection by Toscana virus. METHODS: Sand flies were collected by CDC miniature light traps from seven different localities with an altitude range from 399 m to 1496 m. Synanthropic index was calculated for each sand fly species. The collected female sand flies were grouped in 73 pools, with a maximum of 50 specimens per pool, and submitted to real time PCR for TOSV detection. RESULTS: 8 sand fly species were identified morphologically: 5 of the Phlebotomus genus and 3 of the Sergentomyia genus. Phlebotomus sergenti was the most abundant species comprising of 43,12% of identified sand flies, followed by P. papatasi (18,89%) and P. longicuspis (13,43%). Estimated synanthropic indices for these species were between + 1.1 and + 12.6 suggesting a high preference to anthropogenic environments. A total of 3558 sand fly females were grouped in 73 pools (up to 50 sand flies per pool) for TOSV detection. TOSV was detected in one pool (out of 6 tested) from Lalla Laaziza locality (Chichaoua Province) where P. sergenti was the most abundant sand fly species. CONCLUSION: We reported the TOSV for the first time in a central Morocco, where cutaneous leishmaniasis by L. tropica is endemic. This result has epidemiological importance for both researchers and health authorities to monitor circulation of TOSV and implement a surveillance plan of sand fly-borne phleboviruses in Morocco.

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus.

Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could.

Sand Flies and Pathogens in the Lowlands of Emilia-Romagna (Northern Italy).

Cases of sand fly-borne diseases in the Emilia-Romagna region, such as meningitis caused by Toscana virus and human leishmaniasis, are reported annually through dedicated surveillance systems. Sand flies are abundant in the hilly part of the region, while the lowland is unsuitable habitat for sand flies, which are found in lower numbers in this environment with respect to the hilly areas. In this study, we retrieved sand flies collected during entomological surveillance of the West Nile virus (from 2018 to 2021) to assess their abundance and screen them for the presence of pathogens. Over the four-year period, we collected 3022 sand flies, more than half in 2021. The most abundant sand fly species was Phlebotomus (Ph.) perfiliewi, followed by Ph. perniciosus; while more rarely sampled species were Ph. papatasi, Ph. mascittii and Sergentomyia minuta. Sand flies were collected from the end of May to the end of September. The pattern of distribution of the species is characterized by an abundant number of Ph. perfiliewi in the eastern part of the region, which then falls to almost none in the western part of the region, while Ph. perniciosus seems more uniformly distributed throughout. We tested more than 1500 female sand flies in 54 pools to detect phleboviruses and Leishmania species using different PCR protocols. Toscana virus and Leishmania infantum, both human pathogens, were detected in 5 pools and 7 pools, respectively. We also detected Fermo virus, a phlebovirus uncharacterized in terms of relevance to public health, in 4 pools. We recorded different sand fly abundance in different seasons in Emilia-Romagna. During the season more favorable for sand flies, we also detected pathogens transmitted by these insects. This finding implies a health risk linked to sand fly-borne pathogens in the surveyed area in lowland, despite being considered a less suitable habitat for sand flies with respect to the hilly areas.

Assessment of the Costs Related to West Nile Virus Monitoring in Lombardy Region (Italy) between 2014 and 2018.

In Italy, the West Nile Virus surveillance plan considers a multidisciplinary approach to identify the presence of the virus in the environment (entomological, ornithological, and equine surveillance) and to determine the risk of infections through potentially infected donors (blood and organ donors). The costs associated with the surveillance program for the Lombardy Region between 2014 and 2018 were estimated. The costs of the program were compared with a scenario in which the program was not implemented, requiring individual blood donation nucleic acid amplification tests (NAT) to detect the presence of WNV in human samples throughout the seasonal period of vector presence. Considering the five-year period, the application of the environmental/veterinary surveillance program allowed a reduction in costs incurred in the Lombardy Region of 7.7 million EUR. An integrated surveillance system, including birds, mosquito vectors, and dead-end hosts such as horses and humans, can prevent viral transmission to the human population, as well as anticipate the detection of WNV using NAT in blood and organ donors. The surveillance program within a One Health context has given the possibility to both document the expansion of the endemic area of WNV in northern Italy and avoid most of the NAT-related costs.

Geometric morphometrics and machine learning as tools for the identification of sibling mosquito species of the Maculipennis complex (Anopheles).

Geometric morphometrics allows researchers to use the specific software to quantify and to visualize morphological differences between taxa from insect wings. Our objective was to assess wing geometry to distinguish four Anopheles sibling species of the Maculipennis complex, An. maculipennis s. s., An. daciae sp. inq., An. atroparvus and An. melanoon, found in Northern Italy. We combined the geometric morphometric approach with different machine learning alghorithms: support vector machine (SVM), random forest (RF), artificial neural network (ANN) and an ensemble model (EN). Centroid size was smaller in An. atroparvus than in An. maculipennis s. s. and An. daciae sp. inq. Principal component analysis (PCA) explained only 33% of the total variance and appeared not very useful to discriminate among species, and in particular between An. maculipennis s. s. and An. daciae sp. inq. The performance of four different machine learning alghorithms using procrustes coordinates of wing shape as predictors was evaluated. All models showed ROC-AUC and PRC-AUC values that were higher than the random classifier but the SVM algorithm maximized the most metrics on the test set. The SVM algorithm with radial basis function allowed the correct classification of 83% of An. maculipennis s. s. and 79% of An. daciae sp. inq. ROC-AUC analysis showed that three landmarks, 11, 16 and 15, were the most important procrustes coordinates in mean wing shape comparison between An. maculipennis s. s. and An. daciae sp. inq. The pattern in the three-dimensional space of the most important procrustes coordinates showed a clearer differentiation between the two species than the PCA. Our study demonstrated that machine learning algorithms could be a useful tool combined with the wing geometric morphometric approach.

Serological Evidence of Phleboviruses in Domestic Animals on the Pre-Apennine Hills (Northern Italy).

Phleboviruses are arboviruses transmitted by sand flies, mosquitoes and ticks. Some sand fly-borne phleboviruses cause illnesses in humans, such as the summer fevers caused by the Sicilian and Naples viruses or meningitis caused by the Toscana virus. Indeed, traces of several phleboviral infections have been serologically detected in domestic animals, but their potential pathogenic role in vertebrates other than humans is still unclear, as is the role of vertebrates as potential reservoirs of these viruses. In this study, we report the results of a serological survey performed on domestic animals sampled in Northern Italy, against four phleboviruses isolated from sand flies in the same area. The sera of 23 dogs, 165 sheep and 23 goats were tested with a virus neutralization assay for Toscana virus, Fermo virus, Ponticelli I virus and Ponticelli III virus. Neutralizing antibodies against one or more phleboviruses were detected in four out of 23 dogs, 31 out of 165 sheep and 12 out of 23 goats. This study shows preliminary evidence for the distribution pattern of phleboviral infections in different animal species, highlighting the potential infection of the Toscana virus in dogs and the Fermo virus in goats.

Co-Circulation of Phleboviruses and Leishmania Parasites in Sand Flies from a Single Site in Italy Monitored between 2017 and 2020.

Sand flies transmit Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools (6 Toscana virus positive pools, 2 Corfou virus positive pools, 42 Fermo virus positive pools, and 7 Ponticelli virus positive pools, and 4 unidentified phlebovirus positive pools). Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.