Veterinaria Italiana, 44 (3), 499‐512 First report on entomological field activities for the surveillance of West Nile disease in Italy Luciano Toma(1, 2), Micaela Cipriani(1), Maria Goffredo(1), Roberto Romi(2) & Rossella Lelli(1) Summary Keywords West Nile virus (WNV) is neuropathogenic for Culex pipiens, Italy, Monitoring, Mosquito, birds, horses and humans and is maintained in West Nile, Surveillance, Virus. natural cycles between birds and mosquitoes, particularly the Culex genus; horses and Introduction humans are considered to be incidental hosts. West Nile virus (WNV) is a mosquito‐borne A surveillance plan was implemented in Italy virus that belongs to the family Flaviviridae and in 1998, following a limited outbreak of WNV is a member of the Japanese encephalitis equine encephalomyelitis and a WNV complex. WNV is neuropathogenic for birds, outbreak in France very close to the Italian horses and humans (59) and is maintained in border. This plan to assess the risks of the natural cycles between birds and mosquitoes, virus being introduced again included particularly those of the Culex genus. Horses entomological surveillance performed in and humans are considered to be incidental 15 study areas considered ‘at risk’ of WNV hosts (3). WNV is widely distributed in Africa, introduction in the country. Entomological the Middle East, Eurasia and, was introduced surveys conducted in Italy from 2003 to 2007 into North America more recently (23, 28, 44). resulted in the capture of a total of Human and animal infections were not 28 798 mosquitoes, of which there were documented in the western hemisphere until 14 765 adults and 14 033 larvae belonging to the 1999 outbreak that occurred in the New 22 species. According to the literature, eight of York City metropolitan area. In 2003, WNV the species identified have been found to be activity was reported in 46 states and caused naturally infected with WNV or were illness in over 9 800 people. Since its detection successfully infected in the laboratory in some in the United States in 1999, WNV has spread parts of Europe and in the United States, across 47 mainland states (65), resulting in a namely: Aedes albopictus (Skuse, 1897) total of 27 598 cases with 1 086 deaths in (= Stegomiya albopicta), Aedes vexans (Meigen, humans (7). WNV occurred in Canada in 2001 1830), Anopheles maculipennis Meigen, 1818, (20) and reached the countries of the Caribbean Coquillettidia richiardii (Ficalbi, 1889), Culex and Central America in 2003 (8, 43). It has also modestus Ficalbi, 1889, Culex pipiens Linnaeus, been circulating in several countries in 1758, Culex theileri Theobald, 1903 and continental Europe and in the Mediterranean Ochlerotatus caspius (Pallas, 1771) (= Aedes Basin. Recent outbreaks causing human caspius). encephalitis occurred in Algeria in 1994 (39), in Romania between 1996 and 2000 (62), in the (1) National Reference Centre for Exotic Diseases of Animals (CESME), Entomological Survey for West Nile Disease Surveillance in Italy, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Via Campo Boario, 64100 Teramo, Italy [email protected] (2) Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate (MIPI) (Department of Infectious, Parasitic and Immuno-mediated Diseases), Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy © IZS A&M 2008 www.izs.it/vet_italiana Vol. 44 (3), Vet Ital 499 First report on entomological field activities Luciano Toma, Micaela Cipriani, Maria Goffredo, for the surveillance of West Nile disease in Italy Roberto Romi & Rossella Lelli Czech Republic in 1997 (39), in Russia in 1999 performed using the complement fixation (CF) (26) and in Israel in 2000 (67). Epizootics of the test at the Italian National Centre for Exotic disease in horses occurred in Morocco in 1996 Diseases (CESME, Istituto Zooprofilattico (39), in Italy in 1998 (2) and in France in 2000 Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (39). One of the peculiarities of this flavivirus in Teramo). Subsequently, WNV was isolated is that it can be transmitted by different genera and identified by molecular analysis at the and several species of mosquitoes. To date, the Pasteur Institute in Paris in December 1998 list of mosquitoes in which WNV has been from two of the Italian horses presenting signs isolated includes at least 75 species, even if of encephalitis (2). This event, together with a additional mosquito species have been found more recent outbreak of WNV that occurred positive in the United States (West Nile virus very close to the Italian border in France (38) isolated, West Nile RNA detected or West Nile and the extensive presence in the country of antigen detected using a variety of diagnostic another potential vector, Ae. albopictus (50, 54), tests) (5, 6). WNV is enzootic primarily encouraged the Italian government to amongst birds (27) and appears less frequently implement a surveillance plan to assess the in mammals, with equines and humans risk of the virus being introduced yet again. considered to be dead‐end hosts (13). Culex Since 2002, the Italian Ministry of Health has pipiens is generally regarded to be the most approved a national plan based on serological competent vector of WNV in Europe and was and entomological surveys (involving sentinel possibly involved in the Cerbaie‐Fucecchio chickens and equines in the first case and outbreak (2) and in Romania, where it acted as mosquitoes in the second) by promulgating both enzootic and epizootic vector (55). three ministerial orders and a ministerial Nevertheless, to define the possible role of decree since 2007 November (30, 31, 32, 33). Cx. pipiens in the maintenance and This surveillance plan is implemented by the transmission of arboviruses, the bionomics of CESME in Teramo and is co‐ordinated by the the various mosquito biotypes need to be network of Istituti Zooprofilattici Sperimentali understood (45, 66). located across Italy (which all belong to the This virus has also been detected in other Italian National Health/Veterinary Service) haematophagous arthropods. Amongst others, and by the Unit of Vector Control of the Istituto WNV has been detected in the biting midge Superiore di Sanità for entomological expertise. Culicoides sonorensis Wirth & Jones (Diptera: The authors present the results of the Ceratopogonidae) in the United States (41) and entomological survey that lasted five years and in 10 species of ticks (21) from six genera, covered the entire country. These results are namely: Argas, Ornithodoros (Ixodida: Argasidae), compared to information from the literature. Amblyomma, Dermacentor, Hyalomma, Rhipicephalus The results of this entomological inquiry (Ixodida: Ixodidae) (22, 37). However, the represent part of the ongoing surveillance precise role of these and other haemato‐ system that is focused on improving the phagous arthropods in the transmission of understanding of the ecology of WNV in Italy. WNV requires further investigation. A human epidemic of WNV has been ongoing Materials and methods in the United States since 1999, while limited outbreaks have occurred sporadically in Study areas southern Europe following the introduction of A total of 15 study areas considered ‘at risk’ for the virus by infected birds migrating from WNV introduction in Italy were selected for Africa with the subsequent involvement of longitudinal surveillance (Table I; Fig. 1) on local mosquito populations (40). the basis of features considered most suitable In Italy, a limited focus of WNV equine for virus circulation, such as the simultaneous encephalomyelitis involved 14 race horses at presence of humans, stock farms with horses, nine localities in four provinces of the Toscana migratory birds and mosquitoes. These areas Region (central Italy) in 1998. Diagnosis was were selected and defined in accordance with 500 Vol. 44 (3), Vet Ital www.izs.it/vet_italiana © IZS A&M 2008 Luciano Toma, Micaela Cipriani, Maria Goffredo, First report on entomological field activities Roberto Romi & Rossella Lelli for the surveillance of West Nile disease in Italy standardised criteria developed by the for a total of about 16 surveys per year for five Important Bird Areas (IBA) European years (2003‐2007). Adult mosquitoes were Programme (16) and in collaboration with the collected using Centers for Disease Control Italian National Institute for Wildlife (INFS) in (CDC) light traps and by aspiration in resting Bologna, namely: areas regularly inhabited by sites (mainly animal shelters); all samples were 20 000 or more water birds or by 1% of the stored in dry conditions. A total of 28 light individuals in a population of one species or traps were used, two for each study area; these subspecies of water birds (12). Study areas were placed in shaded positions that were were georeferenced and included in a list of sheltered from wind and rain at a distance of the Italian wetlands used for a waterfowl about 1.5 m from the ground. They were winter census. Italy was divided into a grid of operated from dusk to the early morning. In square units of 400 km2 (20 km each side) by addition, one trap was placed near an animal using geographic information systems (GIS). shelter and another near the henhouse where The extent of the ‘at risk’ areas included all the ‘sentinel’ chickens were stocked for the squares (comprising the monitoring sites) purposes of the serological surveillance. separated by a 20 km buffer drawn around the ‘Sentinel’ refers to cases when an animal (most centroids (15). often a chicken) is kept in a known location Entomological collection procedures (e.g. chicken coop) and its blood is sampled routinely (usually every week or two) and The collection of adult mosquitoes and larvae tested for the presence of WNV. If the test was performed by the staff of the Istituti reveals a positive result, this is an indication Zooprofilattici Sperimentali involved in the that virus is circulating in the area under study Italian national plan of WNV surveillance in (7).