Phlebovirus detection on phlebotomine sandflies in Lampedusa Island (Italy).

Phleboviruses are common human pathogens diffused on the Mediterranean area whose infection can cause the typical prodromal symptom of a mild three­days fever. In particular, Toscana Virus (TOSV) has a great concern since its capacity to provoke central nervous system disorders like meningoencephalitis. Furthermore, as the phlebotomine arthropod vectors represent the main carrier for pathogens of the genus Leishmania as well, the purpose of the study was to investigate the presence of TOSV in Lampedusa, Italy previously reported for leishmaniosis infection cases. The survey was carried out through an initial sampling phase of sand flies, by means of CDC light traps, and a second step of molecular analyses. The genomic S­segment of TOSV was targeted. The positive samples were sequenced and compared with those available in GenBank™ using Basic Local Alignments Tool (BLAST) analyses. The study revealed for the first time the presence of TOSV in Lampedusa, Italy. The entomological studies directed on vectors are currently widely used in sand fly surveillance, and new data on TOSV are of public health concern.

An Epidemiological Study to Investigate Links between Atmospheric Pollution from Farming and SARS-CoV-2 Mortality.

Exposure to atmospheric particulate matter and nitrogen dioxide has been linked to SARS-CoV-2 infection and death. We hypothesized that long-term exposure to farming-related air pollutants might predispose to an increased risk of COVID-19-related death. To test this hypothesis, we performed an ecological study of five Italian Regions (Piedmont, Lombardy, Veneto, Emilia-Romagna and Sicily), linking all-cause mortality by province (administrative entities within regions) to data on atmospheric concentrations of particulate matter (PM2.5 and PM10) and ammonia (NH3), which are mainly produced by agricultural activities. The study outcome was change in all-cause mortality during March-April 2020 compared with March-April 2015-2019 (period). We estimated all-cause mortality rate ratios (MRRs) by multivariate negative binomial regression models adjusting for air temperature, humidity, international import-export, gross domestic product and population density. We documented a 6.9% excess in MRR (proxy for COVID-19 mortality) for each tonne/km2 increase in NH3 emissions, explained by the interaction of the period variable with NH3 exposure, considering all pollutants together. Despite the limitations of the ecological design of the study, following the precautionary principle, we recommend the implementation of public health measures to limit environmental NH3 exposure, particularly while the COVID-19 pandemic continues. Future studies are needed to investigate any causal link between COVID-19 and farming-related pollution.

Genomic Analysis and Lineage Identification of SARS-CoV-2 Strains in Migrants Accessing Europe Through the Libyan Route.

Many African countries, representing the origin of the majority of refugees, asylum-seekers, and other migrants, toward regions bordering on the Mediterranean area, are experiencing sustained local transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sicily is one of the main entry gates of migrants crossing into Europe. We conducted a pilot study, based on the full-genome sequencing of SARS-CoV-2 strains isolated from migrants coming to Sicily by crossing the Mediterranean Sea, with the aim to investigate the viral genome polymorphism and to describe their genetic variations and the phylogenetic relationships. On June 21, a nongovernmental organization vessel rescued 210 migrants crossing the Mediterranean Sea from Libya to Sicily. Of them, 13.4% tested positive for SARS-CoV-2. Eighteen whole genome sequences were obtained to explore viral genetic variability. All but one of the sequences clustered with other viral African strains within the lineage A, whereas only one intermixed among B.1 lineage genomes. Our findings documented that most of the investigated migrants acquired SARS-CoV-2 infection before landing in Sicily. However, SARS-CoV-2 transmission during travel or in overcrowded Libyan immigrant camps and/or illegal transport boats could not be ruled out. SARS-CoV-2 molecular surveillance on migrants arriving in Europe through the Sicilian gate may improve the knowledge of global SARS-CoV-2 transmission dynamic also in light of the emergence of new variants.

Preharvest and Postharvest Applications of a Pomegranate Peel Extract to Control Citrus Fruit Decay During Storage and Shelf Life.

Green and blue molds are the most important postharvest diseases affecting citrus in storage. These diseases are commonly controlled with fungicides, but legislative restrictions, consumer concerns, and the development of resistant strains of the pathogens have increasingly led to the search for alternative methods of control. A pomegranate peel extract (PGE) was very effective in controlling Valencia orange and clementine postharvest rot under commercial conditions. After cold storage and 7 days of shelf life, the incidence of decay on oranges sprayed before harvest with PGE at 12, 6, and 3 g/liter was reduced by 78.9, 76.0, and 64.6%, respectively. Similarly, postharvest dipping treatments with PGE reduced rot by 90.2, 84.3, and 77.6%, respectively. Comparable levels of protection were also achieved on clementines. On both oranges and clementines, the extract provided a significantly higher level of protection compared with imazalil, a fungicide commonly used for postharvest treatments. The high level of efficacy and the consistent results on different fruit species (clementines and oranges) and with different application methods (preharvest and postharvest) were evidence of reliability and flexibility. PGE also showed a strong antimicrobial activity against fungi and bacteria, suggesting its possible use in sanitizers to reduce the microbial contamination of recirculated water in packinghouses. The results of the present study encourage the integration of conventional chemical fungicides and sanitizers with PGE to control citrus postharvest rot.

Development and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves.

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods.

A molecular method to assess Phytophthora diversity in environmental samples.

Current molecular detection methods for the genus Phytophthora are specific to a few key species rather than the whole genus and this is a recognized weakness of protocols for ecological studies and international plant health legislation. In the present study a molecular approach was developed to detect Phytophthora species in soil and water samples using novel sets of genus-specific primers designed against the internal transcribed spacer (ITS) regions. Two different rDNA primer sets were tested: one assay amplified a long product including the ITS1, 5.8S and ITS2 regions (LP) and the other a shorter product including the ITS1 only (SP). Both assays specifically amplified products from Phytophthora species without cross-reaction with the related Pythium s. lato, however the SP assay proved the more sensitive and reliable. The method was validated using woodland soil and stream water from Invergowrie, Scotland. On-site use of a knapsack sprayer and in-line water filters proved more rapid and effective than centrifugation at sampling Phytophthora propagules. A total of 15 different Phytophthora phylotypes were identified which clustered within the reported ITS-clades 1, 2, 3, 6, 7 and 8. The range and type of the sequences detected varied from sample to sample and up to three and five different Phytophthora phylotypes were detected within a single sample of soil or water, respectively. The most frequently detected sequences were related to members of ITS-clade 6 (i.e. P. gonapodyides-like). The new method proved very effective at discriminating multiple species in a given sample and can also detect as yet unknown species. The reported primers and methods will prove valuable for ecological studies, biosecurity and commercial plant, soil or water (e.g. irrigation water) testing as well as the wider metagenomic sampling of this fascinating component of microbial pathogen diversity.