Tick species from Africa by migratory birds: a 3-year study in Italy.

The role of resident or migratory birds in dispersal of tick species and tick-borne pathogens is still poorly known in Italy. We report here the results of a 3-year project based on sampling ticks from migratory birds, as well as from the vegetation at three stop-over sites for migrants, namely the islands of Ventotene (Latium), Asinara (Sardinia) and Ustica (Sicily). During the spring seasons from 2017-2019, in total 2681 ticks were collected, 2344 of which were sampled from migratory birds and 337 from the vegetation. Ticks were identified by morphology or by molecular tools when necessary. In total, 16 tick species were identified among which the following were exclusively found on birds: Hyalomma rufipes (43.3%), Hy. truncatum (0.1%), Ixodes frontalis (11.8%), Ix. inopinatus (0.2%), Ix. ricinus (3%), Haemaphysalis punctata (0.08%), Hae. erinacei (0.1%), Amblyomma variegatum (0.08%) and Argas vulgaris 0.1%), whereas five species were exclusively collected from the vegetation: Rhipicephalus bursa (10.5%), Rh. turanicus (5.9%), Rh. sanguineus sensu lato (2%), Rh. pusillus (2.4%), Hae. sulcata (0.08%). Hy. marginatum (10.3%) and Ix. ventalloi (9.3%) were found both on birds and on the vegetation on the island Ustica. It is worth noting that the search for ticks on the vegetation did not detect allochthonous tick species. Although we found several interesting local species and allochthonous ticks like Hy. rufipes, Am. variegatum and Ar. vulgaris on birds, further investigations are needed to better define the possible role of migratory birds in the introduction of ticks and tick-borne diseases in Italy, above all after the evidence of imported ticks positive to Crimean Congo hemorrhagic fever (CCHF) virus in several European countries.

Trade-Offs with rehabilitation Effectiveness (REs) and Efficiency (REy) in a sample of Italian disabled persons in a in post-acuity rehabilitation unit.

BACKGROUND: Intensive Rehabilitation Centres, known in Italy as "code 56", admit patients who need to recover from an acute episode. Different Rehabilitation Impact Indices have been proposed as composite rehabilitation outcomes measuring the rate of improvement due to a rehabilitation program. The most widely employed measure the performance of Activities of daily living in rehabilitation is the modified Barthel Index. The Barthel Index-based Rehabilitation Impact Indices are the Rehabilitation Effectiveness and the Rehabilitation Efficiency. AIM: The aim of our study was to evaluate the trade-off between Rehabilitation Effectiveness tayand Rehabilitation Efficiency with respect to the Barthel Index admission score and the Length Of Stay, and their ideal ranges that optimized both indices. METHODS: We retrospectively evaluated data of all patients admitted to intensive rehabilitation unit of the Scientific Institute for Research and Healthcare San Raffaele Pisana of Rome, from January 2006 to March 2018. The primary outcome measures of our study were patient's Rehabilitation Effectiveness and Rehabilitation Efficiency during the hospital stay. RESULTS: A database of 3,466 patients was analysed and the Rehabilitation Effectiveness and Rehabilitation Efficiency indexes were calculated. We calculated the median rank ratio of the Rehabilitation Effectiveness to the Rehabilitation Efficiency against Barthel Index scores. We calculated the median rank ratio of the Rehabilitation Effectiveness to Rehabilitation Efficiency against Barthel Index scores and days of stay. The median rank ratio of the Rehabilitation Effectiveness to the Rehabilitation Efficiency value were 1 in the range of Barthel Index scores from 32 to 42. The median rank ratio of the Rehabilitation Effectiveness to Rehabilitation Efficiency value were 1 for a Length of Stay corresponding to 33 days. CONCLUSIONS: In our study we calculated the Trade-offs between Rehabilitation Effectiveness and Rehabilitation Efficiency with respect to admission Barthel Index Score and Length Of Stay in a population of 3,466 patients affected by orthopedic (1,707) and neurological (1,759) diseases. Every member of the healthcare team should be aware of such trade-offs when they make decisions about rehabilitation services.

Colonization of the Mediterranean basin by the vector biting midge species Culicoides imicola: an old story.

Understanding the demographic history and genetic make-up of colonizing species is critical for inferring population sources and colonization routes. This is of main interest for designing accurate control measures in areas newly colonized by vector species of economically important pathogens. The biting midge Culicoides imicola is a major vector of orbiviruses to livestock. Historically, the distribution of this species was limited to the Afrotropical region. Entomological surveys first revealed the presence of C. imicola in the south of the Mediterranean basin by the 1970s. Following recurrent reports of massive bluetongue outbreaks since the 1990s, the presence of the species was confirmed in northern areas. In this study, we addressed the chronology and processes of C. imicola colonization in the Mediterranean basin. We characterized the genetic structure of its populations across Mediterranean and African regions using both mitochondrial and nuclear markers, and combined phylogeographical analyses with population genetics and approximate Bayesian computation. We found a west/east genetic differentiation between populations, occurring both within Africa and within the Mediterranean basin. We demonstrated that three of these groups had experienced demographic expansions in the Pleistocene, probably because of climate changes during this period. Finally, we showed that C. imicola could have colonized the Mediterranean basin in the Late Pleistocene or Early Holocene through a single event of introduction; however, we cannot exclude the hypothesis involving two routes of colonization. Thus, the recent bluetongue outbreaks are not linked to C. imicola colonization event, but rather to biological changes in the vector or the virus.

Schmallenberg virus in Italy: a retrospective survey in Culicoides stored during the bluetongue Italian surveillance program.

Following the first case of Schmallenberg (SBV) in northern Italy in February 2012, virus detection was conducted on midges collected during the national entomological surveillance program for bluetongue (BT). Six cattle farms, within a radius of 50 km from the SBV case, were selected for a 12 month study, aiming to determine when the virus entered the area, if it was capable of overwintering, and the possible role played by each species of the Obsoletus complex in disseminating the infection. A total of 33,724 Culicoides were collected at the six sites between June 2011 and June 2012. Species belonging to the Obsoletus Complex were the most abundant (94.44%) and, within the complex, Culicoides obsoletus was the most prevalent species in the studying area (65.4%). Nearly 7000 Culicoides midges were screened, either in pools or individually, for SBV by real-time RT-PCR. Viral genome was detected in six pools of the Obsoletus complex, collected at three sites between September and November 2011, and in a single parous female of C. obsoletus, collected in May 2012. As a result of the BT surveillance program in Italy it was possible to demonstrate, retrospectively, that SBV has circulated in at least three Italian provinces since early September 2011, nearly 5 months prior and as far as 40 km away from the first detected case. Similarly, the survey confirmed the presence of SBV in the vector population 3 months after the outbreak, following a cold winter during which the blacklight traps failed to catch active adult midges.

A geographical information system-based multicriteria evaluation to map areas at risk for Rift Valley fever vector-borne transmission in Italy.

Rift Valley fever (RVF) is a severe mosquito-borne disease that is caused by a Phlebovirus (Bunyaviridae) and affects domestic ruminants and humans. Recently, its distribution widened, threatening Europe. The probability of the introduction and large-scale spread of Rift Valley fever virus (RVFV) in Europe is low, but localized RVF outbreaks may occur in areas where populations of ruminants and potential vectors are present. In this study, we assumed the introduction of the virus into Italy and focused on the risk of vector-borne transmission of RVFV to three main European potential hosts (cattle, sheep and goats). Five main potential mosquito vectors belonging to the Culex and Aedes genera that are present in Italy were identified in a literature review. We first modelled the geographical distribution of these five species based on expert knowledge and using land cover as a proxy of mosquito presence. The mosquito distribution maps were compared with field mosquito collections from Italy to validate the model. Next, the risk of RVFV transmission was modelled using a multicriteria evaluation (MCE) approach, integrating expert knowledge and the results of a literature review on host sensitivity and vector competence, feeding behaviour and abundance. A sensitivity analysis was performed to assess the robustness of the results with respect to expert choices. The resulting maps include (i) five maps of the vector distribution, (ii) a map of suitable areas for vector-borne transmission of RVFV and (iii) a map of the risk of RVFV vector-borne transmission to sensitive hosts given a viral introduction. Good agreement was found between the modelled presence probability and the observed presence or absence of each vector species. The resulting RVF risk map highlighted strong spatial heterogeneity and could be used to target surveillance. In conclusion, the geographical information system (GIS)-based MCE served as a valuable framework and a flexible tool for mapping the areas at risk of a pathogen that is currently absent from a region.

Endophily in Culicoides associated with BTV-infected cattle in the province of Limburg, south-eastern Netherlands, 2006.

Culicoides were captured at a BTV-infected dairy near Gulpen in the province of Limburg (south-east Netherlands) between 14 September and 4 October 2006. Onderstepoort-type blacklight traps were used to sample Culicoides both inside and outside a partially open shed housing 11 cattle. A total of 28 light trap collections were made at the shed and yielded: 9371 Culicoides representing 11 species; >90% comprised five potential vectors of BTV and in order of abundance were Culicoides obsoletus and Culicoides scoticus (of the Obsoletus Complex), Culicoides dewulfi, Culicoides pulicaris and Culicoides chiopterus; Culicoides imicola, the principal Mediterranean (and African) vector of BTV, was absent. 2339 Culicoides representing seven species were captured inside (endophily) the cattle shed; >95% comprised the Obsoletus Complex and C. dewulfi. Conversely, the Pulicaris Complex, represented by five species and including C. pulicaris, showed strong exophily with >97% captured outside the shed. 7032 Culicoides were captured outside the shed, approximately threefold more than inside. This trend was reversed on an overcast day, when eightfold more Culicoides were captured inside; this indicates that when the light intensity outdoors is low Culicoides will attack (i) earlier in the day while cattle are still at pasture, and (ii) might follow cattle into the sheds in the late afternoon leading to elevated numbers of biting midges being trapped inside the shed during the subsequent hours of darkness. Culicoides were captured inside the shed on all 14 sampling nights. On occasion up to 33% were freshly blood fed indicating they had avidly attacked the cattle inside (endophagy); because half the cattle had seroconverted to BTV, and because no cattle were left outdoors at night, the data indicate that (i) the housing of animals in partially open buildings does not interrupt the transmission of BTV, and/or (ii) BTV is being transmitted while cattle are grazing outdoors during the day. The capture of partially engorged midges inside the shed shows they are being disturbed while feeding; this may lead to cattle being attacked repeatedly, and if these attacks include older parous BTV-infected Culicoides, may enhance virus dissemination (particularly in sheds where cattle stand close together). Endo- and exophagy by potential vector Culicoides--coupled to increased adult longevity and multiple feeding events in single (potentially) infected midges--would ensure an R0 of >1, resulting in the continued maintenance and spread of BTV within local vertebrate populations. Four light trap collections made additionally in a mature deciduous forest 70 m from the shed yielded a high proportion (48%) of gravid females amongst which 10% had incompletely digested blackened blood meals in their abdomens; the absence of this age category in Culicoides captured at the sheds indicates that all Culicoides, after engorgement, exit the buildings to undergo oogenesis elsewhere. In Europe, the blacklight trap is used widely for the nocturnal monitoring of Culicoides; a drawback to this approach is that this trap cannot be used to sample midges that are active during the day. Because diurnal biting in vector Culicoides may constitute a significant and underestimated component of BTV transmission a novel capture methodology will be required in future and is discussed briefly.

Influence of biotic and abiotic factors on the distribution and abundance of Culicoides imicola and the Obsoletus Complex in Italy.

Culicoides imicola Kieffer (Culicoides, Diptera: Ceratopogonidae) is the principal vector of bluetongue virus (BTV) to ruminant livestock in southern Europe. The secondary potential vectors are Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle of the Obsoletus Complex, Culicoides pulicaris (Linnaeus) of the Pulicaris Complex and Culicoides dewulfi Goetghebuer of the subgenus Avaritia Fox. Between 2000 and 2004 >38,000 light-trap collections were made for Culicoides across Italy including the islands of Sardinia and Sicily. Mapping of the 100 largest collections of C. imicola and of the Obsoletus Complex showed them to be disjunct overlapping in only 2% of the 200 municipalities selected. For each municipality the average values were calculated for minimum temperature, aridity index, altitude, terrain slope, normalised difference vegetation index (NDVI) and percentage forest cover. A factor analysis identified two principal factors ('biotic' and 'abiotic') and explained 84% of the total variability; a discriminant analysis classified correctly 87.5% of the observations. The results indicate adult populations of C. imicola to occur in more sparsely vegetated habitats that are exposed to full sunlight, whereas species of the Obsoletus Complex favour a more shaded habitat, with increased green leaf density. Heliophily and umbrophily, by shortening or lengthening the respective adult life cycles of these two vectors, will likely impact on the ability of each to transmit BTV and is discussed in the light of the current outbreak of BTV across the Mediterranean Basin.

Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequences.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages - the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world.

Bluetongue virus isolations from midges belonging to the Obsoletus complex (Culicoides, Diptera: Ceratopogonidae) in Italy.

Between July and September 2002 there were outbreaks of bluetongue on three sheep holdings in the communities of San Gregorio Magno (Salerno, Campania), Laviano (Salerno, Campania) and Carpino (Foggia, Puglia), and the involvement of bluetongue virus (btv) was confirmed serologically and virologically. The mortality rate was at least 11 per cent and involved btv serotype 2 (btv-2) and serotype 9 (btv-9). These holdings were also surveyed for the Culicoides (Diptera: Ceratopogonidae) vectors; approximately 10,000 midges belonging to 15 species were captured, but they did not include a single specimen of the classical Afro-Asiatic bluetongue vector, Culicoides imicola. Species belonging to the Obsoletus complex dominated the light-trap collections, and Culicoides obsoletus Meigen, Culicoides scoticus Downes and Kettle and Culicoides dewulfi Goetghebuer constituted 90 per cent of all the Culicoides species captured. Fifty-six pools of the Obsoletus complex (excluding C dewulfi), each containing 100 individual midges and containing only parous and gravid females, were assayed for virus. btv-2 was isolated from three pools from San Gregorio Magno and Carpino, and btv-9 was isolated from one pool from Laviano. These results indicate that a species other than C imicola is involved in the current re-emergence of bluetongue in the Mediterranean Basin, but whether it is C obsoletus sensu stricto or C scoticus, or both, is uncertain.

The taxonomy of Culicoides vector complexes - unfinished business.

The thirty species of Culicoides biting midges that play a greater or lesser role in the transmission of bluetongue (BT) disease in the pantropical regions of the world are listed. Where known, each species is assigned to its correct subgenus and species complex. In the Mediterranean region there are four species of Culicoides involved in the transmission of BT and belong in the subgenera Avaritia Fox, 1955 (three species) and Culicoides Latreille, 1809 (one species). Using both morphological and molecular second internal transcribed spacer (ITS2) sequence data, the authors reappraise the taxonomy of these four species and their congeners. A total of 56 populations of Culicoides collected from across Italy and representing 17 species (18 including the outgroup taxon C. imicola Kieffer, 1913) were analysed. The findings revealed the following: C. imicola is the only species of the Imicola Complex (subgenus Avaritia) to occur in the Mediterranean region. In Europe the subgenera Avaritia and Culicoides (usually, but not quite correctly, equated with the C. obsoletus and C. pulicaris groups, respectively) are both polyphyletic, each comprising three or more species complexes (including a hitherto unknown complex). About half the species studied could not be identified with certainty; furthermore, the results indicate that at least three previously described species of Palaearctic Culicoides should be resurrected from synonymy. Finally, a high level of taxonomic congruence occurred between the morphological and the molecular data. One of the 'new' vector species, C. pulicaris, was described by the father of taxonomy, Carl Linnaeus, in 1758, but today, almost 250 years later, no monograph has appeared that treats the Culicoides fauna of the northern hemisphere as a whole. At a time when such economically important livestock diseases as BT are affecting ever larger areas of Europe, it would seem appropriate to commence the production of such a monograph to aid in the field identification of vector Culicoides. This 'unfinished business' might best be achieved through a collaborative network embracing all ceratopogonid specialists currently active in both the Palaearctic and Nearctic faunal realms.

Serological evidence of bluetongue and a preliminary entomological study in southern Croatia.

In December 2001, bluetongue (BT) was confirmed serologically by the Croatian Veterinary Institute using the competitive enzyme-linked immunosorbent assay (c-ELISA). Results of the serological testing of blood samples from ruminants in the Dubrovacko-Neretvanska County are presented (3,318 sera of ruminants from 53 herds were examined). In total, 357 bovine sera (178 or 49.9% positive), 1,268 ovine sera (174 or 13.7% positive) and 1,693 caprine sera (270 or 15.9% positive) were tested. Antibodies to BT virus serotype 9 were detected in 212 of the positive sera by serum neutralisation. A preliminary light-trap survey for midges of the Culicoides genus was also performed in the Dubrovacko-Neretvanska County. Fourteen light-trap collections from seven locations were examined and yielded a total of 4,872 Culicoides of which 4,492 (92%) belonged to the Obsoletus Complex (including C. obsoletus and C. scoticus).

Culicoides (Diptera: Ceratopogonidae) in Albania: results of the 2002 entomological survey for bluetongue.

A survey for Culicoides Latreille, 1809, was made in Albania in 2002 to establish whether Culicoides imicola Kieffer, 1913, the main vector of bluetongue virus in the Mediterranean Basin, or any other suspected vector species, was present. The collections and analyses were performed in accordance with the protocols of the National Reference Centre for Exotic Diseases (CESME: Centro Studi Malattie Esotiche) in Teramo, Italy. A total of 43 catches were made in October and November in 15 districts (Bulqise, Devoll, Dibre, Durres, Fier, Gjirokaster, Has, Kolonje, Korce, Librazhd, Permet, Pogradet, Shkoder, Tirane and Tropoje). Twenty species of Culicoides were identified in the collections; the most abundant species belonged to the Obsoletus Complex (98% of total Culicoides in some catches). Culicoides imicola was never captured during the survey. However, a larger number of Culicoides collections and collection sites are needed to exclude the presence of this species at low abundance levels.

Entomological surveillance of bluetongue in Italy: methods of capture, catch analysis and identification of Culicoides biting midges.

To elucidate the epidemiology of vector-borne diseases that can affect livestock in the Mediterranean Basin and elsewhere, it is essential to obtain a clear understanding of the life-cycle and habits of the vector insects involved. One purpose of such investigations is to provide data for an epidemiological surveillance system. As this depends heavily upon the collection of specimens in the field, it is necessary to establish the kinds of information required, and how it can be obtained. This requires, in turn, that the method (and instrument) of capture be standardised, so that all data are as complete as possible, are comparable, and are informative at many levels. Within the surveillance system for bluetongue (BT) in Italy, the National Reference Centre for Exotic Diseases (CESME: Centro Studi Malattie Esotiche) is leading an intensive and countrywide survey for Culicoides (Diptera: Ceratopogonidae) using standardised methods and protocols developed in collaboration with the Onderstepoort Veterinary Institute in South Africa. These methods have now also been implemented outside Italy in Malta, Croatia, Albania and Romania. This system includes the field protocols developed for the collection of Culicoides, the laboratory protocols developed around the insect analyses and the computer-based recording of all field data. Finally, the authors provide an 'Easy key' for the rapid identification of the principal BT vector C. imicola, and for grouping species that belong to the Obsoletus and Pulicaris vector complexes, and to the Nubeculosus and Schultzei potential vector complexes.

Distribution and abundance of Culicoides imicola, Obsoletus Complex and Pulicaris Complex (Diptera: Ceratopogonidae) in Italy.

Between 2000 and 2003, thousands of light-trap collections for Culicoides were made throughout Italy and a detailed distribution map of the primary vector of bluetongue (BT) virus (BTV), C. imicola compiled. In some areas, however, where clinical BT occurred and C. imicola could not be captured, the virus was isolated from biting midges belonging to the Obsoletus and/or the Pulicaris Complexes. Thus, the distribution and abundance of these two species complexes in Italy, as determined from about 3,000 collections, are reported here also and compared to that of C. imicola (from about 24,000 collections). The probable spread of the main vector of BT, C. imicola, into the northern third of Italy, and the widespread prevalence of additional vectors of the Obsoletus and Pulicaris Complexes, indicate nearly all regions of Italy to be at some risk to incursions of BTV. However, these complexes comprise at least six and twelve species, respectively, so precisely which species are able to transmit BTV remains incompletely known.

Entomological surveillance for bluetongue on Malta: first report of Culicoides imicola Kieffer.

A survey for Culicoides Latreille, 1809, was made on Malta in 2002 to establish whether Culicoides imicola Kieffer, 1913, the principal vector of bluetongue virus (BTV) in the Mediterranean Basin, or any other suspected vector species, was present. The collections and analyses were performed in accordance with the protocols of the National Reference Centre for Exotic Diseases (CESME Centro Studi Malattie Esotiche) in Teramo, Italy. Eighty-four catches were made between May and October at six permanent sites, namely: Mellieha, Rabat, San Gwann, Zejtun (Malta), Gharb and Sannat (Gozo island). The traps were placed near cattle (four farms), cattle and sheep (one farm: Rabat) and sheep and goats (one farm: Mellieha). Culicoides midges were found in 91.66% (77/84) of the catches and the highest number of midges per catch was 1 726. Culicoides imicola was confirmed on Malta for the first time in October 2002 and was found at four sites (San Gwann, Sannat, Gharb and Mellieha) but at very low abundance levels (<0.1% of the total Culicoides collected). Culicoides paolae Boorman, 1996 was the most widespread and abundant species (more than 80% of total Culicoides). Midges of the Obsoletus Complex were rare, with less than 10 individuals captured. Other species of Culicoides identified in the collections were: C. submaritimus Dzhafarov, 1962, C. cataneii Clastrier, 1957, C. circumscriptus Kieffer, 1918, C. jumineri Callot and Kremer, 1969, C. kingi Austen, 1912, C. maritimus Kieffer, 1924 and C. newsteadi Austen, 1921.

Laboratory survival and blood feeding response of wild-caught Culicoides obsoletus Complex (Diptera: Ceratopogonidae) through natural and artificial membranes.

In late summer 2002, live wild-caught midges of the Obsoletus Complex were collected using blacklight traps placed at a horse stable in Teramo (Abruzzo, Italy). For the survival study under laboratory conditions, 1,500 Obsoletus Complex midges were kept at 17 degrees C-25 degrees C and provided only with a sucrose solution. Of these, 150 (10%) survived for at least 40 days and 3 midges were still alive after 92 days. In addition, 10 midges survived 10 days at 4 degrees C. For the feeding trials, 40 blood-meals (9,440 midges) were administered, 27 of which were successful (67.5%); the feeding rate ranged from 0.3% to 16.7%, with a total of 592 engorged midges. Similar feeding rates (U Mann-Whitney test=129.5 p>0.05) were obtained when natural (day-old chicken skin) and artificial (stretched parafilm) membranes were used. To infect the insects, a field strain of bluetongue (BT) virus (BTV) serotype 2 isolated from the spleen of a sheep during the 2000 Italian outbreak was added to the blood-meal. Two different viral solutions, with titres of 10(6)TCID(50)/ml and 10(7)TCID(50)/ml, were prepared. Uninfected blood was significantly more appetising (U Mann-Whitney test=88.5 p<0.05) than the infected meal and the midges preferred (U Mann-Whitney test=48 p<0.05) to feed on blood containing BTV-2 at a lower titre. A total of 251 midges were fed on BTV-2 infected blood and were then incubated at 23 degrees C-25 degrees C and fed with a sucrose solution for 10 days. During the incubation period, the dead insects were collected daily and analysed for evidence of virus infection. Of the 251 engorged midges, 54 (21.5%) died in the feeding chambers or during sorting on the chill table, 136 died within the first 10 days and 61 survived longer. BTV was isolated only from those which died just after feeding (52.6%; 10/19) or 24 h later (47.8%; 11/23). Considering the small number of midges tested after 10 days of incubation, the prevalence of infection detected in this study (95% probability) would have been higher than 4.74%. These preliminary results appear very promising as this is the first time that midges of the Obsoletus Complex have been successfully fed under laboratory conditions.

The isolation of bluetongue virus from field populations of the Obsoletus Complex in central Italy.

Between July and September 2002, bluetongue (BT) virus (BTV) serotypes 2 and 9 caused mortalities amongst sheep in the communities of San Gregorio Magno (Salerno, Campania), Laviano (Salerno, Campania) and Carpino (Foggia, Puglia), central Italy. On three of the affected farms, approximately 10,000 specimens of Culicoides were captured, representing fifteen species. Not a single specimen of the classical Afro-Asiatic BT vector, C. imicola Kieffer, was found; species of the Obsoletus Complex dominated the light-trap collections (90%) and included C. obsoletus (Meigen), C. scoticus Downes and Kettle and C. dewulfi Goetghebuer. Fifty-eight pools of the Obsoletus Complex (excluding C. dewulfi), each numbering 100 individuals per pool, and containing only parous and gravid females, were assayed for virus. BTV serotype 2 (BTV-2) was isolated from three pools (San Gregorio and Carpino) and BTV-9 from one (Laviano). These results indicate clearly that a species other than C. imicola is involved in the current re-emergence of BT in the Mediterranean Basin, but whether this is only C. obsoletus sensu stricto, or only C. scoticus, or both together, has yet to be established.

Towards the identification of potential infectious sites for bluetongue in Italy: a spatial analysis approach based on the distribution of Culicoides imicola.

A geographic information system (GIS) based on grids was developed by the National Reference Center for Veterinary Epidemiology at the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZS) in Teramo to identify potential infectious sites for bluetongue (BT) disease in Italy. Geographical and climatic variables were used to build a spatial process model (SPM); the different layers were combined by sequential addition. The final grids (with a cell size of 0.0387 decimal degrees) were generated for each season of the year, and the suitability of each cell for the presence of C. imicola given a value ranking from 0 to 10. While this model more accurately predicts the presence of C. imicola in the Basilicata and Sicily regions, it still over-predicted its presence in the Puglia region. This could be due to the occurrence of calcareous soils which dominate the Puglia landscape. The present SPM is an additive model that assigns an equal weight to each variable. However, the results suggest the existence of hitherto unconsidered variables that significantly influence the prevalence of C. imicola. To reflect their importance, these variables should be assigned a higher weighting in future models. However, the decision in regard to precisely what this weighting should be depends on a very thorough knowledge of the ecology of C. imicola.

Field disinfestation trials against Culicoides in north-west Sardinia.

Bluetongue (BT) first affected Sardinia in August 2000, spreading rapidly across the island causing more than 6,000 outbreaks and significant economic damage. Culicoides imicola Kieffer (Diptera: Ceratopogonidae) was the main vector of the disease and was also found to be the most abundant Culicoides species on Sardinia. During 2002, a field trial was conducted to evaluate the efficacy of an insecticide on local Culicoides populations in north-western Sardinia. A synthetic pyrethroid derivative (Mycrocip, ICF, Cremona, Italy) was used on two farms where outbreaks of BT had been reported; a third farm was used as control. The same treatment was repeated after 15 days. For the collection of Culicoides, two blacklight traps were placed on each farm and operated every second day for two weeks before and after insecticide treatment. Insect collections and data analyses were performed in accordance with the protocols of the Italian National Reference Centre for Exotic Diseases (CESME: Centro Studi Malattie Esotiche). For each collection, the total number of insects, Culicoides spp. and C. imicola was determined. A slight decrease in the number of Culicoides collected on treated farms was recorded for only a few days after treatment. Mycrocip played a secondary role in suppressing insect numbers, but did not reduce the number of Culicoides. Indeed, periodic variations of Culicoides population sizes correlated with significant changes in weather conditions that prevailed, including oscillating temperatures, winds and relative humidity.