Medical and Veterinary Entomology (2009) 23, 202–208
Entry of bluetongue vector Culicoides imicola into livestock premises in Spain C. C A L V E T E1 , R. E S T R A D A2 , M. A. M I R A N D A3 , R. D E L R I O3 , ´ S4 , F. J. B E L D R O N5 , A. M A R T ´I N E Z5 , A. J. C A L V O5 and D. B O R R A J. L U C I E N T E S2 1 Unidad
de Sanidad y Producci´on Animal, Centro de Investigaci´on y Tecnolog´ıa Agroalimentaria (CITA), Gobierno de Arag´on, Zaragoza, Spain, 2 Departamento de Patolog´ıa Animal, Universidad de Zaragoza, Zaragoza, Spain, 3 Laboratorio de Zoolog´ıa, Universidad de las Islas Baleares, Palma de Mallorca, Spain, 4 Instituto de Biolog´ıa Animal de las Islas Baleares (IBABSA), Palma de Mallorca, Spain and 5 Area de Prevenci´on y Control de Epizoot´ıas, Sanidad Animal y Servicios Ganaderos, grupo TRAGSA (TRAGSEGA), Madrid, Spain
Abstract. 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. Key words. Culicoides imicola, African horse sickness, bluetongue, exophilic, Spain.
Introduction Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are the main vectors implicated in the transmission of several arboviruses affecting domestic ruminants and equids. Culicoides imicola is considered a potential vector of diseases such as epizootic haemorrhagic disease (EHD), equine encephalosis (EE), Akabane disease (AKA), bovine ephemeral fever (BEFV), African horse sickness (AHS) and bluetongue disease (BT); the latter two are diseases of international significance. Other Culicoides species have been reported to act
as vectors of African horse sickness virus (AHSV) and bluetongue virus (BTV) worldwide (for review, see Mellor et al., 2000). However, the isolation of both viruses from C. imicola individuals and the coincidence of seasonal and geographic transmission of these viruses and C. imicola abundance indicate that this species is the major field vector of both diseases in many areas, including the Mediterranean basin (Mellor et al., 1983; Mellor, 1996; Mellor & Wittmann, 2002). Observations in South Africa during the 19th and 20th centuries indicated that AHS could be partially prevented by stabling horses from some hours before sunset until a few
Correspondence: Dr C. Calvete, Unidad de Sanidad Animal, Centro de Investigaci´on Tecnolog´ıa Agroalimentaria (CITA-Gobierno de Arag´on), Ctra Monta˜nana 930, 50059 Zaragoza, Spain. Tel: + 34 76 716453; Fax: + 34 76 711 6335; E-mail:
[email protected] © 2009 The Authors
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Journal compilation © 2009 The Royal Entomological Society
Entry of Culicoides imicola into stables 203 hours after sunrise, suggesting that the disease vector was a nocturnal exophilic blood-sucking insect (Paton, 1863; Theiler, 1921). Given that C. imicola is the main AHSV vector in many areas of that country (Meiswinkel et al., 2000), the species has been largely assumed to be exophilic. The stabling of livestock at night has also been recommended to reduce BT transmission in domestic ruminants in Europe (EU Council Directive 2000/75/EC). During the most recent BT epizootics in Spain, caused by BTV-4 and BTV-1, prior confinement in stables for at least 15 days was a mandatory requirement for livestock to be transported. Although the exophilic nature of C. imicola has been widely accepted by animal health managers, knowledge of this behaviour in this species is very limited; only Barnard (1997) and Meiswinkel et al. (2000) have explored the outdoor/indoor preferences of C. imicola. Catches of C. imicola in both these studies were higher outdoors than indoors, supporting the assumed exophilic preference of this species. Results from Barnard (1997) also suggested that, by contrast with the passive entry found with other Culicoides species, C. imicola actively enters stables, probably in search of host animals or a place to hide. Both these studies were performed in South African Culicoides populations and no information exists on the exophilic behaviour of other C. imicola populations. Given this lack of knowledge, data were collected on the indoor and outdoor presence of C. imicola at stables located at several farms during research aimed at evaluating ways to protect livestock from BTV vectors in Spain (Calvete et al., 2007), enabling an assessment, in this study, of the degree of entry of C. imicola into stables in this region.
Materials and methods Culicoides imicola data Culicoides imicola collections were conducted on three farms located in Badajoz province, in southwestern peninsular Spain. The first farm was sampled in November 2006, and the second and third in October 2007. At each site the livestock were permanently confined in stables and most of the manure was removed each morning. Culicoides sampling was carried out using 4-W ultraviolet light traps fitted with a suction fan and a collecting vessel containing a water solution of 33% ethanol and 33% ethylene glycol for sample preservation (miniature blacklight model 1212; John Hock Co., Gainesville, FL, U.S.A.). Traps were placed both inside and outside stables at each farm. Outside traps were hung at a height of 1.75 m on posts located within 15 m of external walls. Inside traps, hung at the same height, were located beyond the reach of livestock and were not visible from the outside. Traps were operated nightly from 1 h before sunset until 1 h after sunrise, at which time any trapped Culicoides species were collected and placed into 70% ethanol until processed in the laboratory, where specimens of C. imicola were identified on the basis of their wing pattern, as described by Rawlings (1996). All captured C. imicola specimens were sexed, and females were age-graded as either nulliparous or parous
according to the method of Dyce (1969). Amongst parous females the occurrence of parous gravid females was also recorded; in the 2007 samples parous engorged females were also noted. Because temperature can limit Culicoides species activity (Mellor et al., 2000) and may influence entry into buildings, the outdoor minimum temperature at each farm was recorded nightly during sampling. A mercury max–min thermometer placed on one of the outside traps was used to record the minimum temperature.
Farms and stables The first farm (the sheep farm) housed 170 sheep in a 40 × 30-m concrete building (the sheep stable) which varied in height from 2.5 m to 3.0 m at the centre. There were 18 openings (each 1.0 × 0.6 m) in the outer wall only, at a height of 1.5 m from the floor. Nine of the openings were permanently closed with glass and the other nine were completely open. In the centre of the building was an open area measuring 28 × 18 m to which access of livestock was restricted by a brick wall 1.5 m high, leaving a continuous opening of 1 × 64 m in the inner wall of the space devoted to housing livestock. Sheep access between the outdoors and the open area was via an outer gate (3.0 × 2.5 m). Access between the open area and the housing space was via three 2.0 × 1.5-m gates. The combined openings amounted to about 92.4 m2 of unimpeded access for host-seeking Culicoides in the outer and inner walls combined. The nearest livestock comprised a ewelamb flock in a separate building 70 m distant on the second farm in the study (see below). Nine light traps were evenly placed close to the outer wall inside the livestock space of the sheep stable, and six traps were placed outside, two in the centre of open ground. Sampling for C. imicola was carried out during five consecutive nights. The second farm (the ewe-lamb farm) housed 150 ewe-lambs in a 40 × 30-m concrete building (the ewe-lamb stable), which varied in height from 2.5 m to 3.0 m in the centre. There were 40 openings each of 1.0 × 0.6 m in the outer wall, at a height of 1.5 m from the floor. Of these, 20 were permanently closed with glass and the remainder were open. The doors of this stable remained closed throughout the sampling period, so the combined openings translated to about 12 m2 of unimpeded access for Culicoides. The nearest livestock (about 100 sheep) were located on the sheep farm (see above). Four traps, two inside and two outside, were used to sample C. imicola abundance during five consecutive nights. The third farm (the calf farm) was 15 km distant from the first and second farms. It included a 10 × 7-m brick building (the calf stable), 3 m in height, which formed part of the dairy. The stable had three windows (1 × 1 m) at 1.5 m from the floor, and these remained open during the sampling period, providing 3 m2 of unimpeded access for Culicoides. This stable housed four calves; the nearest livestock were 51 dairy cows in a shed 35 m distant. Culicoides imicola sampling was carried out during 16 consecutive nights using two inside and two outside traps.
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 202–208
204 C. Calvete et al. The area of unimpeded access has been associated with the degree of entry of C. imicola into stables (Barnard, 1997; Meiswinkel et al., 2000). Given that the three stables differed markedly in structure and size, the ratio of unimpeded to impeded surface area access was calculated for each in order to compare this characteristic among stables. The impeded surface area was estimated from the surface area of the outer (and inner, in the case of the sheep stable) walls minus the unimpeded surface area.
Statistical analyses Variations in the number of C. imicola captured by farm, trap location (inside or outside) and their interactions were fitted to a generalized linear model (GLM) (McCullagh & Nelder, 1997). The interaction term was included to test the influence of stable on the degree of entry (i.e. the inside : outside catch ratio). The daily total numbers of C. imicola captured in traps located inside or outside on each farm were combined, and the mean number caught daily per trap in inside and outside traps was calculated and used as the dependent variable after being log-transformed (log [x + 1.1]). A normal distribution was used as the error distribution. A second GLM was fitted to test the influence of temperature on the degree of entry of C. imicola into stables. In this case the log-transformed daily mean number of C. imicola captured per trap in inside traps was the dependent variable, and the outside minimum temperature on the sampling night was the independent variable. To control for the effect of farm and nightly variations in C. imicola abundance and activity among populations, farm and the daily log-transformed mean number of C. imicola captured in outside traps were also included as predictor variables. Initially the model was adjusted to include the second order interactions between temperature and the other two predictor variables in order to detect differences in the slopes. As none were found, the final model was fitted without interactions. A normal distribution was used as the error distribution. Variations in the proportion of males and the different age-graded groups of females captured as a function of trap location, Culicoides farm population and their interactions were also tested by fitting GLMs. In this case the ratios of males and each female age-graded group to total C. imicola specimens captured were calculated as a function of trap location for each sampling occasion and farm. To fit the models, the ratios were root-square transformed and used as
dependent variables. To analyse the root-transformed ratio for males, a gamma distribution with log-link function was used as the error distribution (McCullagh & Nelder, 1997), whereas root-transformed ratios for nulliparous and parous females (including the gravid and engorged females) were analysed using a normal distribution as the error distribution. Ratios for parous gravid and parous engorged females (the latter only for 2007 data) were also analysed separately using a gamma distribution with log-link function. In all models, the sheep farm Culicoides population and its outside location were used as the control. In the case of models fitted to engorged females, where only data for the calf and ewe-lamb farms were available, the latter were used as the control. Statistical differences were considered significant at P < 0.05.
Results A total of 12 366 Culicoides spp., comprising 10 different species, were collected during the three sampling occasions. Of these, 9493 (76.8%) were C. imicola, of which 2343 (24.7%) were captured in outside traps and 7150 (75.3%) were captured in inside traps. Of the total C. imicola collected, 336 (3.5%) were males, 3307 (34.8%) were nulliparous females and 5850 (61.6%) were parous females; amongst the latter, 121 (1.3%) were gravid. In 2007, 108 engorged females were collected, constituting 1.2% of the total C. imicola specimens captured in the two sampling events performed that year. The overall effects of predictor variables on variation in the daily log-transformed mean number were statistically significant among farm populations (F = 5.70, d.f. = 2, P = 0.006) and between trap locations (F = 9.25, d.f. = 1, P = 0.004), but their interaction was not significant (F = 1.15, d.f. = 2, P = 0.325). The greatest mean catches occurred at the calf farm (parameter ± standard error [SE]) (0.33 ± 0.10; t = 3.35, P = 0.002) and the lowest mean catches occurred at the ewelamb farm (–0.21 ± 0.13; t = −1.69, P = 0.099). Inside traps consistently captured more C. imicola than outside traps at all farms (0.26 ± 0.08; t = 3.04, P = 0.004) (Table 1). The degree of entry into the sheep stable (control) was greater than that for either the ewe-lamb stable (−0.07 ± 0.13; t = −0.57, P = 0.569) or the calf stable (−0.11 ± 0.10; t = −1.07, P = 0.291), but the differences were not significant. The mean inside : outside catch ratio varied between 6.10 and 1.95 among farms (Table 1), and was notably greater than the ratios of unimpeded : impeded access surface area for the three stables.
Table 1. Mean number (standard deviation) per trap/day of catches of Culicoides imicola sampled inside and outside stables at each farm. Inside : outside mean catch ratio, unimpeded access surface area, and unimpeded : impeded access surface area ratio are also presented. n represents the number of trap collections. Inside Farm
n
Sheep Ewe-lamb Calf
45 10 32
Outside n
67.89 (84.62) 34.30 (24.56) 117.22 (163.84)
30 10 32
11.13 (15.72) 8.10 (3.68) 60.06 (59.02)
Catches ratio
Unimpeded access surface, m2
6.1 4.2 1.9
92.4 12 3
Unimpeded access ratio 18.9 × 10−2 3.5 × 10−2 4.3 × 10−2
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 202–208
An apparent direct relationship of mean inside : outside catch ratio with unimpeded access surface area was found for each stable, but not with the unimpeded : impeded access surface area ratio (Table 1). Mean minimum outside temperatures recorded were 12.9 ◦ C (range 7.5 − 16 ◦ C), 12.9 ◦ C (range 1 − 16 ◦ C) and 11.7 ◦ C (range 7 − 14 ◦ C) for the sheep, ewe-lamb and calf farms, respectively. No statistically significant association was found between inside daily mean trap catches and outside minimum temperature during sampling (F = 0.10, d.f. = 1, P = 0.750), and no association was found with farm (F = 1.09, d.f. = 2, P = 0.355) or outside daily mean trap catches (F = 0.45, d.f. = 1, P = 0.508) (Fig. 1). Proportions of males, nulliparous females and engorged females differed significantly among the farm populations of C. imicola (Tables 2 and 3). The proportions of males captured at the calf and ewe-lamb farms were higher (0.39 ± 0.17; W = 5.04, P = 0.025) and lower (−0.61 ± 0.22; W = 7.29, P = 0.007), respectively, than the proportion of males captured at the sheep farm (control). The proportion of nulliparous females was significantly higher (0.10 ± 0.04; t = 2.47, P = 0.017) for the calf farm than for the sheep farm, but lower (although not significantly) for the ewe-lamb farm (−0.10 ± 0.05; t = −1.76, P = 0.084). The proportion of parous females was significantly lower (−0.07 ± 0.04; t = −2.04, P = 0.047) at the calf farm relative to the sheep farm, but higher (although not significantly) at the ewe-lamb farm (0.09 ± 0.05; t = 1.88, P = 0.067). In addition, the proportion
Daily mean trap catches inside
Entry of Culicoides imicola into stables 205 3.0 2.5 2.0 1.5 1.0 0.5 0 0
0.5
1.0
1.5
2.0
2.5
Daily mean trap catches outside Fig. 1. Observed distribution of log-transformed daily mean trap catches of Culicoides imicola inside stables relative to catches outside. , sheep stable; , ewe-lamb stable; •, calf stable.
of engorged females at the calf farm was lower (−0.28 ± 0.13; W = 4.89, P = 0.027) than at the ewe-lamb farm. The proportion of males and engorged females varied significantly according to trap location because a lower proportion of males was captured inside than outside (−0.32 ± 0.15; W = 4.79, P = 0.029), and more engorged females were captured inside (0.60 ± 0.13; W = 22.41, P < 0.001). However, these proportions varied among stables as proportionately fewer males were captured (−0.55 ± 0.23; W = 5.81, P = 0.016) inside at the ewe-lamb stable than at the sheep stable, although there was no significant difference between the calf and
Table 2. Mean daily ratio (standard deviation) of males and each female age-graded group to total Culicoides imicola specimens captured outside and inside stables. Sheep stable
Males Nulliparous females Parous females Gravid females Engorged females
Ewe-lamb stable
Calf stable
Inside
Outside
Inside
Outside
Inside
Outside
0.04 0.17 0.79 0.01
0.01 0.38 0.60 0.05
0 (0) 0.14 (0.07) 0.86 (0.07) 0.01 (0.005) 0.03 (0.02)
0.03 0.21 0.76 0.07 0
0.01 0.44 0.54 0.02 0.02
0.10 0.33 0.58 0.07 0.01
(0.04) (0.11) (0.13) (0.01) –
(0.02) (0.37) (0.36) (0.11) –
(0.05) (0.20) (0.21) (0.09) (0)
(0.02) (0.21) (0.21) (0.03) (0.02)
(0.16) (0.24) (0.25) (0.16) (0.02)
Table 3. Overall effects of generalized linear models fitted to males and each female age-graded group to total Culicoides imicola captured ratios (root-transformed) estimated separately for inside and outside daily catches. P < 0.05 values are in bold.
Males Nulliparous females Parous females Gravid females Engorged females∗ ∗ Model
Culicoides population
Trap location
Stable
W = 8.77, d.f. = 2 P = 0.012 F = 3.37, d.f. = 2 P = 0.043 F = 2.72, d.f. = 2 P = 0.077 W = 0.95, d.f. = 2 P = 0.620 W = 4.89, d.f. = 1 P = 0.027
W = 4.79, d.f. = 1 P = 0.029 F = 0.12, d.f. = 1 P = 0.730 F = 1.52, d.f. = 1 P = 0.224 W = 2.19, d.f. = 1 P = 0.139 W = 22.41, d.f. = 1 P < 0.001
W = 10.57, d.f. = 2 P = 0.005 F = 2.21, d.f. = 2 P = 0.121 F = 1.34, d.f. = 2 P = 0.271 W = 0.11, d.f. = 2 P = 0.946 W = 9.53, d.f. = 1 P = 0.002
fitted using only 2007 data.
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 202–208
206 C. Calvete et al. sheep stables (−0.17 ± 0.17; W = 0.92, P = 0.338). In addition, proportionately more engorged females (0.39 ± 0.13; W = 9.53, P = 0.002) were captured inside the ewe-lamb stable than the calf stable.
Discussion An exophilic tendency in C. imicola species has been suggested on the basis of reports that larger numbers (two- to 10-fold more) were captured outside rather than inside stables (Barnard, 1997; Meiswinkel et al., 2000). By direct contrast, in the current survey higher numbers of C. imicola were detected inside stables than outside (two- to six-fold more). In this survey, the mean number of C. imicola captured per inside trap during all sampling periods at each farm was directly associated with the mean number captured per outside trap (Table 1). Assuming that mean outside catches were directly related to the mean population abundance of C. imicola, it can be concluded that the number of C. imicola entering into the stables was directly related to population abundance outside (in agreement with the two previous reports), but no daily correlation between inside and outside mean trap catches was observed (by contrast with previous reports). Temperature modulates the activity of C. imicola (Mellor et al., 2000), but it does not influence the ratio of entry of C. imicola into stables as this variable affects the catch both inside and outside (Meiswinkel et al., 2000). However, high wind speed alone or in combination with rain can increase the inside : outside catch ratio because the outside catch decreases but the inside catch is unaffected (Barnard, 1997; Meiswinkel et al., 2000). In the current study, the absence of a relationship between daily inside trap catches and minimum temperature, after controlling for daily outside catches, is in agreement with previous reports. The absence of a daily correlation between daily inside and outside catches may be the result of the effect of wind speed or other climatic parameters not controlled for in this study, other than minimum outside temperature. Despite this, the daily correlation of catches in both previous studies was markedly higher than in the current survey, suggesting that in the present study factors other than outside climatic conditions or outside C. imicola abundance were modulating the relationship between inside and outside daily catches. For example, conditioning of the inside–outside temperature gradient by the presence of animals may have influenced the movement of C. imicola. Previous studies have found a direct relationship between the ratio of entry into stables and the surface area of unimpeded access. These studies involved comparisons within the same buildings (Meiswinkel et al., 2000), or between stables of the same size (Barnard, 1997), so comparisons were performed directly between the ratio of catches and the unimpeded surface area. In the current study, the stables differed substantially in size, so the inside : outside catch ratio was compared with the ratio of unimpeded : impeded access surface area. Neither ratio showed a clear direct relationship, mainly because of values for the calf farm. Nevertheless, a direct (although not proportional) relationship was observed between the inside : outside catch
ratio and the size of the unimpeded access surface area, suggesting that (in agreement with previous studies) the degree of entry of C. imicola into stables may also have been modulated by this factor. Despite this apparent relationship, however, the interaction term of the fitted model was not statistically significant (i.e. the model did not detect any statistically significant influence of stable on the degree of entry of C. imicola). Moreover, the marked differences among farms, such as the presence of an open area in the sheep stable, the presence of other livestock housed in nearby sheds (e.g. dairy cows near the calf stable), or variation in attraction into stables secondary to housing different livestock species (Mands et al., 2004) may have strongly influenced the entry of C. imicola (Anderson et al., 1993; Barnard, 1997; Meiswinkel et al., 2000). As a consequence, the apparent agreement with previous studies with respect to the relationship between degree of entry and unimpeded access surface area of stables should be treated with caution because it may have been significantly affected by variables not controlled in our study, such as, among others, the possible different levels of attraction of inside and outside traps for different subpopulations of the Culicoides population (i.e. active host-seeking females) conditioned by the absence or presence of animals in the neighbourhood of traps. The proportions of each sex and female age-graded groups also differed among populations. This variation was mainly a result of the high proportion of males and nulliparous females in the calf farm population and the high proportion of parous females in the ewe-lamb farm population. This variation was not surprising as a logical asynchrony of population dynamics secondary to local and temporal variations in bioclimatic factors was expected. Interestingly, variation in the proportions of males and engorged females as a function of trap location and farm was also observed. The proportion of males was higher outside than inside except for at the sheep stable, where this relationship was reversed. The higher catches of males outside may reflect the fact that they are less attracted to animals than are females, which probably enter seeking hosts. The reason for the greater proportion of males observed inside at the sheep stable is not clear, but may be related to structural differences among the stables, such as the presence of open ground or the proximity of a suitable breeding site for C. imicola (Meiswinkel et al., 2000). Higher proportions of engorged females were captured inside than outside at both stables sampled in 2007. The difference was larger for the ewe-lamb stable as no engorged females were captured outside. This result contrasts with the findings of Barnard (1997), who reported higher catches of engorged females outside than inside. As Barnard (1997) suggested, it may be that the interior of stables provides an easily accessible and secure place for engorged females to hide before they relocate to suitable breeding sites, and that females in search of a place to hide may be less attracted to light traps. However, although our results may partially reflect the cause espoused by Barnard (1997), they also suggest that engorged females may be those that fed on housed livestock, and that the higher proportion of engorged females captured outside the calf
© 2009 The Authors Journal compilation © 2009 The Royal Entomological Society, Medical and Veterinary Entomology, 23, 202–208
Entry of Culicoides imicola into stables 207 stable reflected the presence nearby of dairy cows available as additional and easily accessible (housed in sheds) hosts. The results of the current study call into question the assumed strong exophilic tendencies of C. imicola as they, conversely, may suggest endophilic behaviour of the species in this study area and there is no obvious explanation for the difference. The current study was performed after the usual population peak of C. imicola (Ortega et al., 1998; Miranda et al., 2004), when the outside temperature was declining. One explanation may be that C. imicola is endophilic at the end of its annual biological cycle, when temperatures inside stables are more suitable for activity than those outside. However, no correlation was found between outside minimum temperatures and the degree of entry into stables and, moreover, it is not known if C. imicola is exophilic during the earlier phase of its biological cycle in the study area. However, the maximum temperatures on the sampling nights ranged from 20◦ C to 32◦ C and were compatible with outside activity of the species, and it may be that outside temperature conditions were not sufficiently unsuitable to allow for the detection of thermal preferences in C. imicola and their effects on degree of entry during the study. It is also possible that differences exist in exophilic/endophilic preferences among Culicoides populations, as has been reported for other Diptera species (Kanojia & Geevarghese, 2004). Both previous studies involved South African Culicoides populations, whereas the present study was of Iberian populations. A third, more convincing possibility is that, given the relatively large openings in the stables and the high number of animals inside the stables coupled with the fact that there were no animals on the outside (except at the calf farm, where the lowest catch ratio was estimated), the current results reflect a strong mammophilic tendency in C. imicola (rather than endophilic behaviour) that could override the apparently inherent exophilic behaviour previously reported. This possibility is supported by the fact that engorged females were only collected inside the stables. Additional research, therefore, will be necessary to clarify and explain the entry tendencies of C. imicola in the study area. Although the factors underlying the entry behaviour of C. imicola into stables remain unclear, the results of this study suggest that sheds, stables and other farm premises may not protect livestock from C. imicola in this area, at least during the period of the year when the study was performed. This is emphasized by the fact that most C. imicola captured inside were parous females (i.e. the only form of the species that can transmit the virus as transversal transmission does not occur). The amount of entry into stables reported in the present survey contradicts the accepted view that stabling provides partial protection against the vector activity of C. imicola. However, it remains to be determined whether the amount of entry reported here would occur when inside and outside trapping are based on equal quantities of animal bait. If the entry of C. imicola into stables was driven by mamophilic rather than endophilic behaviour, then, if animals were present on the outside of these stables, more Culicoides midges would probably be collected outside than inside stables. It would be useful to clarify whether the reported protection of stabled livestock is therefore associated with the presence of other
hosts outside because, in fact, a marked reduction in the entry of C. imicola into stables has been reported under these conditions (Anderson et al., 1993; Barnard, 1997; Meiswinkel et al., 2000). Under this assumption stabling will therefore reduce the biting rate and will still be beneficial in the integrated control of bluetongue. However, it is essential that these stables are evaluated and that the factors that attract and govern the entrance of Culicoides species into these stables are determined. Irrespective of whether the studied populations of C. imicola are endophilic for all or part of their annual cycle or whether they are simply strongly mammophilic, our results raise the possibility that, as has been reported for other Culicoides species (Olsson et al., 2007), C. imicola may overwinter in stables. This would have implications for BT and/or AHS epidemiology in areas including southern Europe and the Mediterranean basin.
Acknowledgements Funding for this study was provided by Animal Health and Livestock Services, TRAGSA group (TRAGSEGA) for the project ‘Assessment of the Efficacy of Different Methods of Bluetongue Prevention and of Controlling its Vectors’. We thank A. Boluda, M. D´ıaz-Molina and C. D´ıez-De la Varga for their assistance in fieldwork. Special thanks go to R. Calero for granting permission to work on the Selection and Breeding Animal Centre (CENSYRA) property in Badajoz and for assistance, and to two anonymous referees for commenting on this manuscript.
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