D. Borrás

Spatial distribution of Culicoides imicola, the main vector of bluetongue virus, in Spain

BLUETONGUE (BT) is a disease caused by a virus of the genus Orbivirus that is mainly transmitted among vertebrate hosts by several species of biting midges of the genus Culicoides (Diptera: Ceratopogonidae) (Du Toit 1944, Mellor and Pitzolis 1979). BT virus (BTV) can replicate in all species of ruminants and causes severe disease in certain breeds of sheep and some species of deer (Taylor 1986, MacLachlan 1994). In Europe, the main vector for BTV is Culicoides imicola Kieffer (Mellor 1996), although species included within the Culicoides obsoletus group have been proposed as the probable vector of BTV in some European areas (Mellor and Wittmann 2002, Savini and others 2003, De Liberato and others 2005). Historically, BTV has made only brief, periodic incursions into Europe (Manso-Ribeiro and others 1957, Mellor and Pitzolis 1979, Jennings and others 1983). However, a large BT epidemic, which started in 1998, has affected many European countries, including several that had not previously experienced the virus, and is still ongoing in some areas. This extension of BTV into Europe appears to be partially linked to the recent northward expansion of C imicola across the Mediterranean European countries – an expansion that seems to be a consequence of recent changes in the European climate (Purse and others 2005). In Spain, during the last BT epidemic, BTV entered the Balearic Islands from eastern Europe in 2003. In late 2004, further incursions occurred from Morocco into south-west Spain, and in late 2005 BTV was spreading northwards in peninsular Spain. Previous studies suggested that the distribution of C imicola in Spain was mainly restricted to southwest peninsular Spain, and the Toledo province was thought to be the northernmost site for the species (Ortega and Holbroock 1994, Ortega and others 1997, 1998, Rawlings and others 1997). In the past five years, however, local surveys have reported, for the first time, the presence of C imicola in the Balearic Islands (Miranda and others 2003), in coastal areas of Catalonia (Sarto i Monteys and others 2003), in Madrid and in the eastern coastal provinces of Alicante and Murcia (Martinez 2004; F. Collantes, personal communication). A national surveillance programme aimed at estimating the spatial distribution of C imicola in Spain was carried out during 2004. The landmass of peninsular Spain was divided into 202 quadrats of 50 x 50 km; 100 evenly distributed quadrats were selected as sampling units, and within each sampling unit at least two livestock holdings or farms were sampled. In addition, seven farms were sampled in the Balearic Islands (three on Majorca, three on Minorca and one on Ibiza), five in the Canary Islands (two each on Tenerife and Fuerteventura and one on Gran Canaria), and one in the town of Ceuta, a Spanish town in northern Africa. Two samplings were carried out: one in spring/summer (April to June) and another in summer/autumn (September to November). Each farm was sampled for two consecutive nights. A 4 W UV light trap fitted with a suction fan (Miniature Blacklight model 1212; John Hock) was placed outside each selected farm but within 30 m of livestock, at between 1·7 and 2·5 m above ground level. The traps were lit from just before dusk until after dawn over both sampling days. Specimens of C imicola were identified on the basis of their wing pattern, as described by Rawlings (1996). In approximately one-quarter of the sampling units more than two farms were sampled; most extra samplings occurred during the summer/autumn period. In total, 364 farms were sampled; 229 during spring/summer and 292 in summer/ autumn; 159 of the farms were sampled in both periods. A total of 12,878 C imicola were trapped over the course of both sampling periods. The species was found on all the farms sampled in the Balearic Islands, with the exception of the one on Ibiza; however, it is known that C imicola is present on Ibiza, albeit in very low numbers (B. V. Purse, M. A. Miranda, personal communication). No C imicola were captured on the farm sampled in Ceuta, and the species was not detected in the Canary Islands. C imicola was found in all the provinces of peninsular Spain where it had previously been reported (Fig 1) except Sevilla, where it was not detected on any of the farms sampled. In addition, C imicola was found for the first time in nine other provinces, mainly located to the north and east of the previously defined distribution area of the species. The highest numbers of C imicola trapped in one sampling session of two consecutive nights in provinces where the species had previously been reported were in Malaga (6304 specimens), Huelva (973 specimens) and Jaen (348 specimens). Among the nine provinces where C imicola was recorded for the first time, the highest catches were in Albacete (480 specimens), Avila (194 specimens) and Granada (66 specimens). All of the sampling sessions with the highest catches occurred during the summer/autumn sampling period. The altitudes of the sampled farms ranged from 5 to 1400 m above sea level. The highest altitude at which C imicola was Short Communications

Protection of livestock against bluetongue virus vector Culicoides imicola using insecticide-treated netting in open areas.

The protection of livestock against Culicoides species (Diptera: Ceratopogonidae) using physical barriers or chemically treated barriers is difficult owing to the small size of these biting midges and animal welfare concerns associated with the reduction of air flow. Culicoides imicola Kieffer is the main bluetongue virus vector in the Mediterranean basin, including the southern Iberian peninsula, where livestock is mainly housed in open pens or sheds which offer no physical protection against C. imicola. In this study we assessed the efficacy of surrounding yearling ewe pens with a canvas barrier or a cypermethrin‐treated canvas barrier in reducing the entry of Culicoides spp. and C. imicola. Analyses were based on comparisons of Culicoides catches in traps in pens with and without barriers, and in traps located outside pens. Although there was no clear reduction in the abundance of Culicoides other than C. imicola in pens with either barrier, the C. imicola presence was markedly reduced by the insecticide‐treated barrier compared with the untreated barrier; the latter did not reduce the abundance of this species in pens. Estimates of the protection conferred against C. imicola by the treated barrier differed depending on whether catch comparisons were based on outside traps or on traps located inside no‐barrier pens. The results suggest that the use of insecticide‐treated barriers may reduce contact between livestock and C. imicola in open areas or sheds. More research is necessary to assess the degree of protection as a function of barrier height, C. imicola abundance, and the size of the area to be protected.

Collection of Culicoides spp. with four light trap models during different seasons in the Balearic Islands

Bluetongue (BT) is a viral disease that affects ruminants, being especially pathogenic in certain breeds of sheep. Its viral agent (bluetongue virus; BTV) is transmitted by several species of Culicoides biting midges (Diptera: Ceratopogonidae). Different models of suction light traps are being used in a number of countries for the collection of BTV vector species. To determine the relative effectiveness of different light traps under field conditions, four traps (Onderstepoort, Mini-CDC, Rieb and Pirbright) were compared. These traps were rotated between four sites on a cattle farm in Mallorca (Balearic Islands, Spain) for several non-consecutive nights. Results showed remarkable disparities in the efficacy of the traps for the collection of Culicoides midges. The highest number of midges collected in the Onderstepoort trap (x¯±SD=62±94.2) was not significantly different from that collected in the Mini-CDC (x¯±SD=58±139.2). The Rieb trap collected the lowest number of midges (x¯±SD=3±4.0). Significantly higher mean numbers of midges were collected in the Onderstepoort than in either the Pirbright (P=0.002) or Rieb traps (P=0.008). There were also differences in the Culicoides species composition as determine with the various traps. These results indicate that the Onderstepoort or Mini-CDC traps will be more effective than either the Rieb or Pirbright traps for the collection of large numbers of Culicoides midges.

Entry of bluetongue vector Culicoides imicola into livestock premises in Spain

Culicoides imicola Kieffer is considered to be the main vector of bluetongue disease (BT) and African horse sickness (AHS) in the Mediterranean basin. It has been assumed that this midge species is exophilic and, consequently, that stabling of livestock should provide effective protection against these diseases. This study presents the results of sampling surveys for C. imicola carried out both inside and outside stables on three farms in mainland Spain. The number of C. imicola captured varied as a function of the populations sampled and trap location (inside vs. outside). The daily mean number captured inside during the sampling of each farm population was directly correlated with the daily mean number captured outside, but daily correlation of captures was not observed. By contrast with previous studies, the mean catch of C. imicola inside was consistently higher than that outside. No clear effect of stable characteristics on the degree of entry was detected. In addition, proportions of males and age‐graded female groups varied among populations and with trap location. Proportionately more males and fewer engorged females were captured outside than inside, although the proportions varied among stables. These results contrast with those of previous studies, and with the assumed pronounced exophilic behaviour of C. imicola, and raise important questions about the vector activity of this species in the study area and its implications for the epidemiology of BT and/or AHS.

Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): Results from an international ring trial targeting four species of the subgenus Avaritia

BackgroundBiting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly.MethodsIn this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe.ResultsA total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction.ConclusionsThe results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Variations in the mitochondrial cytochrome c oxidase subunit I gene indicate northward expanding populations of Culicoides imicola in Spain

Culicoides imicola is the main vector for bluetongue (BT) and African horse sickness (AHS) viruses in the Mediterranean basin and in southern Europe. In this study, we analysed partial mitochondrial cytochrome c oxidase subunit I (COI) gene to characterize and confirm population expansion of Culicoides imicola across Spain. The data were analysed at two hierarchical levels to test the relationship between C. imicola haplotypes in Spain (n = 215 from 58 different locations) and worldwide (n = 277). We found nineteen different haplotypes within the Spanish population, including 11 new haplotypes. No matrilineal subdivision was found within the Spanish population, while western and eastern Mediterranean C. imicola populations were very structured. These findings were further supported by median networks and mismatch haplotype distributions. Median networks demonstrated that the haplotypes we observed in the western Mediterranean region were closely related with one another, creating a clear star-like phylogeny separated only by a single mutation from eastern haplotypes. The two, genetically distinct, sources of C. imicola in the Mediterranean basin, thus, were confirmed. This type of star-like population structure centred around the most frequent haplotype is best explained by rapid expansion. Furthermore, the proposed northern expansion was also supported by the statistically negative Tajimas D and Fus Fs values, as well as predicted mismatch distributions of sudden and spatially expanding populations. Our results thus indicated that C. imicola population expansion was a rapid and recent phenomenon.

The influence of sheep age group on the seasonal prevalence of oestrosis in the island of Majorca

Oestrosis is highly prevalent in Mediterranean countries. Understanding the life cycle of Oestrus ovis is crucial to design effective control measures of this myiasis, largely based on the use of macrocyclic lactones. We carried out a survey of ovine oestrosis in the island of Majorca (Spain) and found that 46.03% of animals were infested in a 13-month period. Interestingly, we found significant differences in oestrosis prevalences in winter and autumn when separating the animals by group of age (P<0.001). Pearson correlation analysis showed that prevalence in lambs younger than 4 months was significantly affected by changes in air temperature (P<0.05), but this association was not significant in adult sheep (P=0.081). Chronic infestations or unsystematic treatments may explain confusing results in adult sheep. Observing the evolution of the disease in young lambs, we determined that the hypobiotic period took place from October to February and the beginning of fly activity occurred between May and June. Interannual variations in oestrosis prevalence indicate the need of monitoring the disease to establish the appropriate timing of treatments. We hypothesize that lambs are better indicators of the seasonality of oestrosis than their older counterparts. Furthermore, we propose that observing O. ovis infestations in young lambs can be used as an efficient early warning system of fly activity, to be applied in future control programs.

Modelling the range expansion of the Tiger mosquito in a Mediterranean Island accounting for imperfect detection

BackgroundsAedes albopictus (Diptera; Culicidae) is a highly invasive mosquito species and a competent vector of several arboviral diseases that have spread rapidly throughout the world. Prevalence and patterns of dispersal of the mosquito are of central importance for an effective control of the species. We used site-occupancy models accounting for false negative detections to estimate the prevalence, the turnover, the movement pattern and the growth rate in the number of sites occupied by the mosquito in 17 localities throughout Mallorca Island.ResultsSite-occupancy probability increased from 0.35 in the 2012, year of first reported observation of the species, to 0.89 in 2015. Despite a steady increase in mosquito presence, the extinction probability was generally high indicating a high turnover in the occupied sites. We considered two site-dependent covariates, namely the distance from the point of first observation and the estimated yearly occupancy rate in the neighborhood, as predicted by diffusion models. Results suggested that mosquito distribution during the first year was consistent with what predicted by simple diffusion models, but was not consistent with the diffusion model in subsequent years when it was similar to those expected from leapfrog dispersal events.ConclusionsAssuming a single initial colonization event, the spread of Ae. albopictus in Mallorca followed two distinct phases, an early one consistent with diffusion movements and a second consistent with long distance, ‘leapfrog’, movements. The colonization of the island was fast, with ~90% of the sites estimated to be occupied 3 years after the colonization. The fast spread was likely to have occurred through vectors related to human mobility such as cars or other vehicles. Surveillance and management actions near the introduction point would only be effective during the early steps of the colonization.