Human and entomological surveillance of Toscana virus in the Emilia-Romagna region, Italy, 2010 to 2012.

Toscana virus (TOSV), transmitted by phlebotomine sandflies, is recognised as one of the most important causes of viral meningitis in summer in Mediterranean countries. A surveillance plan based on both human and entomological surveys was started in 2010 in the Emilia-Romagna region, Italy. Clinical samples from patients with neurological manifestations were collected during 2010 to 2012. The surveillance protocol was improved during these years, allowing the detection of 65 human infections. Most of these infections were recorded in hilly areas, where sandflies reach the highest density. Entomological sampling around the homes of the patients resulted in a low number of captured sandflies, while later sampling in a hilly area with high number of human cases (n=21) resulted in a larger number of captured sandflies. Using this approach, 25,653 sandflies were sampled, of which there were 21,157 females, which were sorted into 287 pools. TOSV RNA was detected by real-time PCR in 33 of the pools. The results highlighted the role of Phlebotomus perfiliewi as the main vector of TOSV and a potential link between vector density and virus circulation. This integrated system shows that an interdisciplinary approach improves the sensitiveness and effectiveness of health surveillance.

The experience of West Nile virus integrated surveillance system in the Emilia-Romagna region: five years of implementation, Italy, 2009 to 2013.

Predicting West Nile virus (WNV) circulation and the risk of WNV epidemics is difficult due to complex interactions of multiple factors involved. Surveillance systems that timely detect virus activity in targeted areas, and allow evidence-based risk assessments may therefore be necessary. Since 2009, a system integrating environmental (mosquitoes and birds) and human surveillance has been implemented and progressively improved in the Emilia-Romagna region, Italy. The objective is to increase knowledge of WNV circulation and to reduce the probability of virus transmission via blood, tissue and organ donation. As of 2013, the system has shown highly satisfactory results in terms of early detection capacity (the environmental surveillance component allowed detection of WNV circulation 3­4 weeks before human cases of West Nile neuroinvasive disease (WNND) occurred), sensitivity (capacity to detect virus circulation even at the enzootic level) and area specificity (capacity to indicate the spatial distribution of the risk for WNND). Strong correlations were observed between the vector index values and the number of human WNND cases registered at the province level. Taking into consideration two scenarios of surveillance, the first with environmental surveillance and the second without, the total costs for the period from 2009 to 2013 were reduced when environmental surveillance was considered (EUR 2.093 million for the first scenario vs EUR 2.560 million for the second). Environmental surveillance helped to reduce costs by enabling a more targeted blood unit testing strategy. The inclusion of environmental surveillance also increased the efficiency of detecting infected blood units and further allowed evidence-based adoption of preventative public health measures.

West Nile virus circulation in Emilia-Romagna, Italy: the integrated surveillance system 2009.

Following a large West Nile virus (WNV) epidemic in northeastern Italy in 2008, human and animal surveillance activities were implemented in Emilia Romagna. Human surveillance was performed by serology or genome detection on blood and cerebrospinal fluid for all suspected cases suffering from acute meningoencephalitis in the regional territory. Animal surveillance consisted of passive and active surveillance of horses and active surveillance of wild birds and mosquitoes. Between 15 June and 31 October 2009, nine of 78 possible cases of West Nile neuroinvasive disease were confirmed (three fatal). From May to October, 26 cases of neurological West Nile disease were confirmed among 46 horses. The overall incidence of seroconversion among horses in 2009 was 13%. In 2009, 44 of 1,218 wild birds yielded positive PCR results for WNV infection. The planned veterinary and entomological surveillance actions detected WNV activity from the end of July 2009, about 2-3 weeks before the onset of the first human neurological case. Passive surveillance of horses seems to be an early and suitable tool for the detection of WNV activity, but it will be less sensitive in the future, because an intensive programme of horse vaccination started in June 2009.

A possible role for activated complement component 3 in phagocytic activity exhibited by the mouse trophoblast.

PROBLEM: To determine whether any blood plasma factor may play a regulatory role in trophoblast phagocytosis in rodent early pregnancy. METHOD OF STUDY: The effects of alloplasma on the phagocytosis of cultured mouse trophoblast cells (TCs) were evaluated using erythrocytes as target cells, in the presence of 10% fresh, normal plasma; 10% heat-inactivated plasma; 10% component 3 (C3)-depleted plasma; or medium alone. The possible activation of C3 complement, the phagocytosis of zymosan bound or unbound to C3b, and immunoreactivity to C3b receptor were also estimated. Phagocytic activity was expressed as the percentage of phagocytic TCs, and as the number of phagosomes/TCs. RESULTS: The use of complement sufficient plasma significantly enhanced the phagocytosis of the TCs while the use of heat-inactivated plasma eliminated the erythrophagocytosis. Very low levels of phagocytic activity were seen when the plasma was C3-complement deficient. Phagocytosis of C3b-bound zymosan was remarkable in comparison to zymosan alone, and immunoreactivity to C3b-receptors was seen on the TCs. CONCLUSION: These results indicate the participation of thermosensitive molecules mediating the phagocytosis of TCs and suggest, as in macrophages, a role for C3-C3b in this process.

Differentiation-dependent expression of gelatinase B/matrix metalloproteinase-9 in trophoblast cells.

The purpose of this study was to evaluate the Rcho-1 trophoblast culture system as a model for studying trophoblast invasion and to examine stage-specific expression of enzyme(s) potentially participating in rat trophoblast giant cell invasive behavior. The invasive behavior of the differentiating Rcho-1 trophoblast cells was demonstrated using Matrigel invasion chambers. Gelatin zymography and Western blot analysis of conditioned medium from differentiating Rcho-1 trophoblast cell cultures and rat ectoplacental cone outgrowths revealed a differentiation-dependent increase in gelatinase B/matrix metalloproteinase (MMP-9). Nothern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of Rcho-1 trophoblast or ectoplacental cone cells also showed increasing expression of MMP-9 accompanying cell differentiation. Rcho-1 trophoblast cells stably transfected with MMP-9 promoter/luciferase reporter constructs exhibited a differentiation-dependent increase in MMP-9 promoter activation. In conclusion, trophoblast giant cell differentiation is characterized by transcriptional activation of the MMP-9 gene and appearance of the invasive phenotype.

Induction of erythrophagocytic activity in cultured mouse trophoblast cells by phorbol myristate acetate and all-trans-retinal.

Phorbol myristate acetate (PMA) and all-trans-retinal (retinal) were evaluated as possible phagocytic stimulators of cultured, post implantation, trophoblast cells. Ectoplacental cones dissected from 7.5 day-old mouse embryos provided the source of trophoblastic cells. Co-cultures were performed using stimulated and non-stimulated trophoblast cells and erythrocytes under standard conditions. Phagocytic activity was expressed as the total number of phagocytic cells per ectoplacental cone, and as phagosomic vacuoles per trophoblast giant cell, either in the presence or absence of the stimulators. Both chemical agents had similar effects, less than 12 h after stimulation, statistically significant numbers of erythrophagosomes appear in the trophoblast giant cells (TGC). These findings demonstrate that TGC, like neutrophils and macrophages, can be activated to phagocytosis by exogenous factors. This enhanced activity may result from the generation and release of reactive oxygen species. In conclusion, our data suggest that, because stimulation was provided, the remarkable in vivo phagocytic activity of the trophoblast can be maintained under in vitro conditions, allowing study of the pathways and regulatory steps involved in this process.