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Leishmania (L.) infantum is one of the main causative agents of animal and human leishmaniasis across many endemic areas in South America, Europe, North Africa, and Asia. Despite its clinical significance, little is known about the genetic diversity of L. infantum circulating in a given endemic area. Here, we investigate this important open question by applying a comparative genomics approach to seven L. infantum isolates from different hosts and Italian regions, including the northern part of the country (Emilia-Romagna, RER), Sicily, and Sardinia, as an initial attempt to explore the breadth of parasite genetic heterogeneity in Italy. Additionally, microsatellite analysis was carried out to compare the isolates from RER with other 70 L. infantum strains from the same region as well as 65 strains belonging to the L. donovani complex from other countries. We revealed important karyotypic instability and identified strain-specific changes in gene dosage, which affected important virulence factors such as amastins and surface antigen-like proteins. Single nucleotide polymorphism-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters within the Mediterranean basin and uncovered three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling, these hybrid isolates are representative of a sub-population of parasites circulating in northern Italy that preferentially infect humans but not dogs. Our results place Italy at the crossroads of L. infantum infection in the Mediterranean and call attention to the public health risk represented by the introduction of non-European Leishmania species.IMPORTANCEThis study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum/L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.

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BACKGROUND: Sporadic intracranial vascular malformations can pose significant risk to parturients, and additional reports of management may inform patient care. Here we describe the peripartum management of parturients with intracranial vascular malformations. METHODS: After Institutional Review Board approval, we performed a retrospective analysis of parturients with a known sporadic intracranial vascular malformation including cavernous malformation, developmental venous anomaly, or arteriovenous malformation who delivered at our institution between 2007 and 2020. RESULTS: We identified 10 parturients (five cavernous malformations, three developmental venous anomalies, and two arteriovenous malformations) with 16 deliveries. Among all deliveries, 13 (81.3%) were cesarean deliveries without trial of labor; 11 of these (84.6%) received a single-shot spinal and two (15.4%) received an epidural for surgical anesthesia. Two deliveries (12.5%) began with attempted trial of labor but ultimately required cesarean delivery for failure to progress; one of these cases received epidural anesthesia and the other received combined spinal-epidural anesthesia. One delivery was via spontaneous vaginal delivery with epidural analgesia. Overall, our study's cesarean delivery rate was 93.8% and spontaneous vaginal delivery rate was 6.2%. Three of 16 pregnancies were complicated by seizure, obstructive hydrocephalus, or intracranial hemorrhage. There were no intensive care unit admissions or maternal deaths. CONCLUSIONS: In our case series of 16 deliveries, there were no complications directly resulting from neuraxial procedures. It remains unclear whether intracranial developmental venous anomalies or unruptured arteriovenous malformations impart increased risk during pregnancy. Antepartum planning with a multidisciplinary team approach enables risk stratification and optimal management.

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Solid-phase sorption is widely used for the analysis of gaseous specimens as it allows at the same time to preconcentrate target analytes and store samples for relatively long periods. The addition of internal standards (ISs) in the analytical workflow can greatly reduce the variability of the analyses and improve the reliability of the protocols. In this work, we describe the development and testing of a portable system for the reliable production of gaseous mixture of8D-Toluene in a 1L Silonite canister as well as its reproducible loading into solid-phase sorbing tools as ISs. The portable system was tested using needle trap microextraction, solid-phase extraction, and thin-film microextraction techniques commonly employed for the analysis of gaseous samples. Even though our specific interest is in breath analysis, the system can also be used for the collection of any kind of gaseous specimen. A microcontroller allows the fine control of the sampling flow by a digital mass flow controller. Flow rate and sample volume could be set either through a rotary encoder mounted onto the control board or through a dedicated android app. The variability of the airflow is in the range 5-200 ml min-1and it is lower than 1%, whereas the variability of the IS (8D-Toluene) concentration dispensed over time by the loader measured by selected-ion flow-tube mass spectrometry (MS) is <3%. This combination resulted in intra- and inter-day precision of the amount loaded in the sorbent tools lower than 15%. No carry-over was detected in the loader after the delivery of the8D-Toluene measured by gas chromatography-MS. The8D-Toluene concentration in the canister was stable for up to three weeks at room temperature.

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Visual cues such as plate size, amount of food served and packaging are known to influence the effects of portion size on food intake. Unit bias is a well characterised heuristic and helps to determine consumption norms. In an obesogenic environment where large portions are common place, the unit or segmentation bias may be overridden promoting overconsumption of both amorphous or unit foods. The aim of this review was to investigate the impact of offering unit or amorphous food on the portion size effect (PSE) in children aged 2-12 years. A systematic search for literature was conducted in Medline, PsycInfo and Web of Science in February 2018. A total of 1197 papers were retrieved following the searches. Twenty-one papers were included in the systematic review, of which 15 provided requisite statistical information for inclusion in a random effects meta-analysis. Increasing children's food portion size by 51-100% led to a significant increase in intake (SMD = 0.47, 95% CI: 0.39-0.55). There was no evidence to suggest that increases in consumption were related to food type (p = 0.33), child age (p = 0.47) or initial portion size served (p=0.14). Residual heterogeneity was not significant (p=0.24). The PSE was demonstrated in children aged 2-12 years when offered both unit and amorphous food items. The effect was not restricted by food type, child age or influenced by initial portion size served. Of the studies included in the meta-analysis between study heterogeneity was low suggesting minimal variation in treatment effects between studies, however, more research is required to understand the mechanisms of the PSE in preschool children. Future research should determine feasible methods to downsize portion sizes served to children.

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Hyaluronan (HA) is central in joint and cartilage functions and to restore synovial fluid viscosity. In patients with osteoarthritis (OA), molecular weight (MW) and concentration of hyaluronic acid (HA) are reduced, diminishing joint lubrication. IL-1ß treatment was used to mimic osteoarthritis in a chondrocytes based in vitro model. The aim of our research, using this model and human chondrocytes was to assess the anti-inflammatory effect of H/L-HA hybrid complexes (SINOVIAL-HL®) in comparison with HA at high (H-HA) and low molecular weight (L-HA) separately used, through the evaluation of specific biomarkers involved in cartilage degradation and correlated to osteoarthritis. Specifically, TNF-α and IL-6 mRNA were evaluated by qRT-PCR. Cytokines levels were measured using Bio-plex assays and COMP-2 through immunofluorescence staining and western blot. H/L-HA significantly reduced inflammation biomarkers respect to both L-HA or H-HA separately considered at transcriptional and protein level.

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Short tandem repeats are used as an effective method to trace DNA markers in genotyping. Using a standardized kit, we tested 11 microsatellite markers recommended by the International Society for Animal Genetics (ISAG) in a sample of 495 Sicilian cattle. The aim of this study was to investigate the allele frequencies in the Sicilian cattle population to provide a reference database and at the same time to assess the use of the ISAG microsatellite panel for pedigree analysis. DNA samples were collected from blood and amplified in an 11-plex polymerase chain reaction (PCR); PCR products were injected in a 3130 Genetic Analyzer. All loci showed high mean polymorphism information content (0.768), and the observed mean heterozygosity was less than the expected value (0.732 vs 0.794, respectively). The exact test for Hardy-Weinberg proportions, allele number, and inbreeding coefficient were calculated. Our results indicated that equilibrium was not always maintained. The observed mean homozygote value exceeded the expected value (132.81 vs 102.14), but no evidence for allele dropout was found. These results could be explained by a non-random mating; further studies using a larger number of animals could confirm or invalidate this hypothesis. The probability of identity and exclusion of a locus were also estimated and proved to be useful in paternity testing. The ISAG microsatellite panel is useful to screen the Sicilian bovine kinship. Currently, an allele frequency database is being constructed.

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Different approaches are being developed to improve the differentiation of Leishmania genus using biochemical and molecular methods. In this study, 11 independent polymorphic microsatellites were used for the typing of strains of L. infantum isolated in Sicily. Polymerase chain reaction was employed to amplify the microsatellites contained in 12 DNA regions selected from among more investigated loci. A total of 51 isolates of L. infantum from dogs were tested by using the same locus panel. The products were successively analysed using an automatic sequence detector (ABI PRISM 3130 AB), to discover relevant microsatellite polymorphisms. It was possible to discriminate between MON-1 and non-MON-1 groups. Moreover, the method permitted to distinguish various genotypes of L. infantum isolates within each zymodema. Model- and distance-based analyses of the data set showed comparable results. The frequency of heterozygosity in the alleles analysed varied extremely between the different groups of isolates. As the method exhibits a high level of discrimination, it is suitable for characterization of closely related strains in epidemiological studies.

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Seven microsatellite markers were used to study genetic diversity of three Egyptian (Egyptian Baladi, Barki and Zaraibi) and two Italian (Maltese and Montefalcone) goat breeds. The microsatellites showed a high polymorphic information content (PIC) of more than 0.5 in most of the locus-breed combinations and indicated that the loci were useful in assessing within- and between-breed variability of domestic goat (Capra hircus). The expected heterozygosity of the breeds varied from 0.670 to 0.792. In the geographically wider distributed Egyptian Baladi breed there were indications for deviations from random breeding. Analysis of genetic distances and population structure grouped the three Egyptian goat breeds together, and separated them from the two Italian breeds. The studied Mediterranean breeds sampled from African and European populations seem to have differentiated from each other with only little genetic exchange between the geographically isolated populations.

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A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP)-derived markers) was obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.

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Leishmaniasis is a typical example of a worldwide diffused zoonosis. Geographic distribution depends on the presence of sand fly vectors and animal reservoirs. In Southern Europe, canines are considered the main reservoir of infection, and the phlebotomines are the vectors. In Sicily, as in all Mediterranean areas, sand flies are present almost all year around because the climate permits an uninterrupted lifecycle for the vectors. Visceral leishmaniasis is becoming a real public health concern especially in endemic areas; in fact, it is an opportunistic infection in immunocompromised patients and in HIV-positive subjects. In Italy, the visceral form of the disease is due exclusively to Leishmania infantum ZMON1, and its prevalence is growing. We have developed a highly accurate, reproducible, and sensible real-time polymerase chain reaction (PCR) assay. In a procedure that used a specific couple of primers, a 117-bp fragment was amplified from minicircle kinetoplast DNA (kDNA). The assay was able to detect even a single parasite (200 fg of DNA). In fact, a single parasite contains hundreds of kinetoplast minicircles for each class. We applied a rapid extraction method coupled with the real-time PCR assay. It was not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA, but also more rapid. The assay is useful for the diagnosis of leishmaniasis in dogs and humans, and it facilitates the monitoring of parasite levels during pharmacological treatment.

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Parasitic gastroenteritis is one of the major causes of productivity loss in sheep and goats. This report records two studies of the helminth fauna from post-mortem examination. The first study, performed on the digestive tract of 72 sheep from a central part of Sicily in a high hill village (1,360 meters above sea level), between April 1996 and March 1997, showed an infection rate of 78%. The second study targeted goats from the western part of Sicily and showed an infection rate of 90%. For sheep, a total of 23 species of helminths were identified belonging to the family of Trichostrongyloidea, with the genera Haemonchus, Ostertagia (Teladorsagia), Trichostrongylus, Cooperia, and Nematodirus; Strongilolidea with the genera Oesophagostomum and Chabertia: Ancylostomidea with Bunostomum; and Tricuridea with Tricuris. Teladorsagia circumcincta was the most common in the sheep abomasum, Bunostomum trigonocephalum and Trichostrongylus spp. in the small intestine, and Chabertia ovina and Trichuris ovis in the large intestine. For goats, a total of 12 species were isolated in the abomasum with Teladorsagia circumcincta and Trichostrongylus axei the most common species. In the small intestine, five species were isolated and Trichostrongylus capricola was the dominant species. T. ovis and O. venulosum were dominant in large intestine and in the cecum. We also found species belonging to other ruminants such as O. ostertagi (in cattle) and S. kolchida and O. leptospicularis (in wild ruminants).

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Parasitic egg production was studied in sheep and goats affected by parasitic gastroenteritis. The herds studied were located at different altitudes and in different climatic conditions. Samples were taken every month and the number of eggs per grams of feces was calculated. Observation of preliminary data shows that the maximum peak of egg production was during the winter period, whereas in other countries winter is a period of hypobiosis. This study shows that understanding peak time of infection related to different climatic and environmental conditions will help improve anthelminthic treatments and animal health strategies.

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