During the late summer of 1998, veterinary authorities in Tuscany, Italy, received reports of cases of neurologic disease among horses residing in a large wetland area located in the provinces of Florence and Pistoia. West Nile virus was isolated from two of the six horses that died or were euthanized. A retrospective epidemiologic study identified 14 clinical neurologic cases that occurred from August 20 to October 6 (attack rate of 2.8%). A serologic survey conducted over a 700-km2 area in stables with and without apparent clinical cases confirmed a wider spread of the infection, with an overall seroprevalence rate of 38% in the affected area. No significant differences in age-specific prevalence were observed, suggesting that the horses residing in the area had not been exposed previously to West Nile virus and supporting the hypothesis of its introduction in the wetland area during the first half of 1998.

A two year study (2008-2009) was carried out to monitor the Usutu virus (USUV) circulation in Italy. Sentinel horses and chickens, wild birds and mosquitoes were sampled and tested for the presence of USUV and USUV antibodies within the WND National Surveillance plan. Seroconversion evidenced in sentinel animals proved that in these two years the virus has circulated in Tuscany, Emilia Romagna, Veneto and Friuli Venezia Giulia regions. In Veneto USUV caused a severe blackbird die-off disease involving at least a thousand birds. Eleven viral strains were detected in organs of 9 blackbirds (52.9%) and two magpies (0.5%) originating from Veneto and Emilia Romagna regions. USUV was also detected in a pool of Culex pipiens caught in Tuscany. According to the alignment of the NS5 partial sequences, no differences between the Italian USUV strains isolated from Veneto, Friuli and Emilia Romagna regions were observed. The Italian North Eastern strain sequences were identical to those of the strain detected in the brain of a human patient and shared a high similarity with the isolates from Vienna and Budapest. Conversely, there were few differences between the Italian strains which circulated in the North Eastern regions and the USUV strain detected in a pool of C. pipiens caught in Tuscany. A high degree of similarity at both nucleotide and amino acid level was also found when the full genome sequence of the Italian North Eastern isolate was compared with that of the strains circulating in Europe. The North Eastern Italian strain sequence exhibited 97% identity to the South African reference strain SAAR-1776. The deduced amino acid sequences of the Italian strain differed by 10 and 11 amino-acids from the Budapest and Vienna strains, respectively, and by 28 from the SAAR-1776 strain. According to this study two strains of USUVs are likely to have circulated in Italy between 2008 and 2009. They have developed strategies of adaptation and evolution to spread into new areas and to become established.

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A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 104-105 CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.