Epidemiology of West Nile disease in Europe and in the Mediterranean Basin from 2009 to 2013.

West Nile virus (WNV) transmission has been confirmed in the last four years in Europe and in the Mediterranean Basin. An increasing concern towards West Nile disease (WND) has been observed due to the high number of human and animal cases reported in these areas confirming the importance of this zoonosis. A new epidemiological scenario is currently emerging: although new introductions of the virus from abroad are always possible, confirming the epidemiological role played by migratory birds, the infection endemisation in some European territories today is a reality supported by the constant reoccurrence of the same strains across years in the same geographical areas. Despite the WND reoccurrence in the Old World, the overwintering mechanisms are not well known, and the role of local resident birds or mosquitoes in this context is poorly understood. A recent new epidemiological scenario is the spread of lineage 2 strain across European and Mediterranean countries in regions where lineage 1 strain is still circulating creating favourable conditions for genetic reassortments and emergence of new strains. This paper summarizes the main epidemiological findings on WNV occurrence in Europe and in the Mediterranean Basin from 2009 to 2013, considering potential future spread patterns.

Variability of the hemocyte parameters of cultivated mussel Mytilus galloprovincialis (Lmk 1819) in Sabaudia (Latina, Italy) coastal lagoon.

The Sabaudia's lake consists of a protected coastal lagoon, located in the central Italy, historically characterized by recurrent mortality events of marine fauna during warmer months. A field study was monthly conducted on mussels Mytilus galloprovincialis cultivated inside the lagoon, measuring hemocyte parameters as total circulating count (THC), viability (HV), spreading and oxidative response to in vitro phagocytosis stimulation. A depression of the immune response was observed during the spring season, as indicated by higher values of hemocyte circularity and lower luminescence levels related to respiratory burst, also associated to modulation of THC and HV. The water temperature and the oxygen concentration appeared as the major environmental factors having influence on the phagocytosis activity. Therefore, the hemocyte variations have been intended as early danger signal to evaluate the immunodepression induced by the environmental stressors which could reveal in advance the development of critical situations for mussel survival.

Effects of high temperature and exposure to air on mussel (Mytilus galloprovincialis, Lmk 1819) hemocyte phagocytosis: modulation of spreading and oxidative response.

Hemocytes are a critical component of the mussel defense system and the present study aims at investigating their spreading and oxidative properties during phagocytosis under in vivo experimental stress conditions. The spreading ability was measured by an automated cell analyzer on the basis of the circularity, a parameter corresponding to the hemocyte roundness. The oxidative activity was investigated by micromethod assay, measuring the respiratory burst as expression of the fluorescence generated by the oxidation of specific probe. Following the application of high temperature and exposure to air, there was evidence of negative modulation of spreading and oxidative response, as revealed by a cell roundness increase and fluorescence generation decrease. Therefore, the fall of respiratory burst appeared as matched with the inhibition of hemocyte morphological activation, suggesting a potential depression of the phagocytosis process and confirming the application of the circularity parameter as potential stress marker, both in experimental and field studies.

Age related properties of the Adriatic clam Chamelea gallina (L. 1758) hemocytes.

The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.

Electrochemical genosensors for the detection of Bonamia parasite. Selection of single strand-DNA (ssDNA) probes by simulation of the secondary structure folding.

A post-PCR nucleic acid work by comparing experimental data, from electrochemical genosensors, and bioinformatics data, derived from the simulation of the secondary structure folding and prediction of hybridisation reaction, was carried out in order to rationalize the selection of ssDNA probes for the detection of two Bonamia species, B. exitiosa and B. ostreae, parasites of Ostrea edulis. Six ssDNA probes (from 11 to 25 bases in length, 2 thiolated and 4 biotinylated) were selected within different regions of B. ostreae and B. exitiosa PCR amplicons (300 and 304 bases, respectively) with the aim to discriminate between these parasite species. ssDNA amplicons and probes were analyzed separately using the "Mfold Web Server" simulating the secondary structure folding behaviour. The hybridisation of amplicon-probe was predicted by means of "Dinamelt Web Server". The results were evaluated considering the number of hydrogen bonds broken and formed in the simulated folding and hybridisation process, variance in gaps for each sequence and number of available bases. In the experimental part, thermally denatured PCR products were captured at the sensor interface via sandwich hybridisation with surface-tethered probes (thiolated probes) and biotinylated signalling probes. A convergence between analytical signals and simulated results was observed, indicating the possibility to use bioinformatic data for ssDNA probes selection to be incorporated in genosensors.