Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe

Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap network for the collection of Culicoides, was implemented in 2002 in southern mainland France. The morphological identification of Culicoides can be both tedious and time-consuming because its size ranges from 1.5 to 3 mm. Therefore, an ITS1 rDNA polymerase chain reaction (PCR)-based diagnostic assay was developed to rapidly and reliably identify Culicoides spp. and C. imicola. The aim of this work was to set up a rapid test for the detection of C. imicola amongst a pool of insects collected in areas at risk for BT. The sequence similarity of the rDNA (nuclear ribosomal DNA), which is greater within species than between species, is the foundation of its utilisation in species-diagnostic assays. The alignment of the 11 ITS1 sequences of Culicoides obtained from Genbank and EMBL databases helped us to identify one region in the 5' end and one in the 3' end that appear highly conserved. PCR primers were designed within these regions to amplify genus-specific fragments. In order to set up a C. imicola-specific PCR, another forward primer was designed and used in combination with the previously designed reverse primer. These primers proved to be highly specific and sensitive and permitted a rapid diagnostic separation of C. imicola from Culicoides spp.     

Simultaneous quantification of the relative abundance of species complex members: application to Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae), potential vectors of bluetongue virus

The two sympatric sibling species Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle (Diptera: Ceratopogonidae), are known to be competent vectors for bluetongue virus in the Palaearctic region. However, morphological identification of constituent species is only readily applicable to adult males and these two species distinguishing traits have overlapping character states. As their vector competence may differ in space and time, the correct identification and quantification of specimens of both species are essential for understanding bluetongue epidemiology. However, no molecular tools are available for high-throughput identification of the two species. We therefore developed a quantitative duplex real-time PCR assay to determine the relative abundance of each sibling species in a sample using TaqMan probes. For each species, standard curves were constructed from serial dilutions of purified plasmid DNA containing ITS1-5.8S-ITS2 (rDNA) in the range of 10(-1) to 10(-5)ng/μL. Standard curves were used to quantify samples of mixed C. obsoletus/C. scoticus specimens. Specificity was evaluated with 5156 specimens representing 62 species. Based on the DNA quantities detected according to the standard curves, a quadratic model developed on 1100 males and validated on 555 females was able to predict the relative abundance of each species simultaneously in a one-shot reaction (Pearson coefficient of 0.999). Our assay showed a requirement of two specimens or less for 95% of the predictions, making it highly applicable to field collections. Extensive use of this real-time PCR assay will provide a better understanding of geographical distribution, dynamics, and bionomics on a species level, which is essential for risk assessment. This approach is an important contribution to medical entomology for investigating the vector role of arthropod sibling species.     

Molecular characterization of Swiss Ceratopogonidae (Diptera) and evaluation of real-time PCR assays for the identification of Culicoides biting midges

Biting midges of the genus Culicoides (Diptera, Ceratopogonidae) are vectors of several viruses of veterinary relevance, and they can cause insect bite hypersensitivity. As the morphological identification of these tiny insects is a difficult task in many cases, alternative approaches are expedient. With the aim to develop real-time PCRs, we determined partial mitochondrial cytochrome oxidase I gene (mt COI) sequences from 380 Culicoides midges representing three regions of Switzerland, namely the Alps, Midland north of the Alps (Atlantic climate), and South of the Alps (Mediterranean climate). The same region was also sequenced from non-biting midges of the genera Atrichopogon, Brachypogon, Dasyhelea, Forcipomyia and Serromyia. A total of 21 Culicoides species were identified by morphology. Sequence variability (haplotypes) was observed in all species. For each of C. grisescens and C. obsoletus, a novel cryptic species was identified. Whereas all individuals of C. grisescens and of the cryptic C. obsoletus species (O2) originated only from Alpine sites, the known C. obsoletus (O1) species was found in all three regions. Further, a sister taxon to C. pulicaris was identified based on the mt COI sequences and named Culicoides sp. Alignments of available mtCOI sequences from Ceratopogonidae (GenBank, this study) were used to design real-time PCR primers and probes to distinguish C. chiopterus, C. deltus, C. dewulfi, C. grisescens (including the cryptic species), C. imicola, C. lupicaris, C. obsoletus O1, C. obsoletus O2, C. pulicaris, C. scoticus and Culicoides sp. Specificities of primers and probes was tested with cloned targets representing 1 to 4 haplotypes of 18 Culicoides spp. and 1 haplotype each from 4 other Ceratopogonidae. No cross-reactivity was observed when plasmid template representing 5 × 10(6) gene copies was tested, but it was evident (Ct values ≤ 30) in few instances when plasmid template representing 5 × 10(9) gene copies was utilized, the latter corresponding to the total gene copy number (as determined in this study) in 20 insects. The sensitivities of two assays (C. imicola, C. grisescens) were tested by spiking single insects into pools of 99 or 999, randomly selected non-target Ceratopogonidae (with approx. 90% Culicoides specimens). In the pools of 100, Ct values were in the range of those obtained with single insects when employing 1% of the isolated DNA, whereas the sensitivity with the pools of 1000 was low, presumably due to the low DNA concentrations obtained with a protocol that seems inadequate for these larger pools. Thus, the assays as described are applicable for the specific identification of biting midges in small pools. Primers and probes of this study were devised to be suitable for multiplexed assays but these evaluations await to be performed.     

Comparison of black and white light for collecting Culicoides imicola and other livestock-associated Culicoides species in South Africa

Comparison of the effectiveness of 8W fluorescent black and white light sources, in two 4x4 Latin squares (16 replicates) designs under South African conditions, showed black light to be up to three time more effective in collecting Culicoides imicola Kieffer (Diptera, Ceratopogonidae) and other South African Culicoides species. Four Culicoides species, which were collected in low numbers with black light, were not collected in traps equipped with the white light source. No significant difference was found in the parous rate of the C. imicola populations as determined by the two light sources. The study highlighted the superiority of black light as a preferred collection method for C. imicola, considered to be the most widespread and abundant vector of livestock orbiviruses. The results underline the need to develop and adopt standard techniques for measuring the variables of vectorial capacity.     

A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast? EvaGreen(?) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.     

PCR amplification and sequencing of ITS1 rDNA of Culicoides arakawae

The first internal transcribed spacer (ITS1) of nuclear ribosomal DNA from Culicoides arakawae was amplified by PCR, cloned and sequenced. The wDNAsis software was used to analyze the ITS1 sequences of C. arakawae and other nine species of Culicoides, which were obtained from GenBank and EMBL databases. For all species, the lengths of the ITS1 were 316-469 bp, and the G+C contents were 26.79-34.53%. Based on the lengths of the ITS1 sequences, the 10 Culicoides species could be divided into two groups. The first group consisted of C. arakawae, C. albicans, C. cubitalis, C. pulicaris and C. punctatus, and the second group comprised C. impunctatus, C. nubeculosus, C. variipennis, C. grisescens and C. imicola. The lengths for the first group were 316-347 bp and the second group were 457-469 bp. C. arakawae belonged to the first group by its ITS1 sequence length. Sequence analysis revealed that C. arakawae was genetically more similar to the first group than it was to the second group, consistent with results based on sequence length. The alignment of ITS1 (the alignment length was 500 bp including the gaps) sequences showed that there was a highly conserved region, which was between 288 and 388 bp, except for a few insertions and substitutions. These findings have important implications for the molecular identification of C. arakawae, for studying its molecular genetics and epidemiology, and for studying the molecular systematics of Culicoides.     

Phenology and attraction of potential Culicoides vectors of bluetongue virus in Basque Country (northern Spain)

Bluetongue virus is transmitted by Culicoides biting midges (Diptera: Ceratopogonidae). Culicoides associated with livestock were captured using CDC blacklight traps at three BTV-infected farms in Basque Country between November 2007 and December 2008. Twenty-seven and nineteen Culicoides species were collected in outdoor and indoor habitats respectively. Indoor insect community represented 86.1% of the whole captured individual biting midges. Culicoides obsoletus/Culicoides scoticus (two sibling species of the Obsoletus complex) were dominant throughout all months and sexes with maximum phenological peaks in November 2007 and June-July 2008. Culicoides lupicaris was the second most dominant species followed by Culicoides pulicaris (both species of the Pulicaris complex). Few specimens of Culicoides imicola, the principal Afro-Mediterranean vector of BTV, as well as four new species recorded for the Iberian Peninsula, were also collected. BTV was detected by RT-PCR from pools of C. obsoletus/C. scoticus, C. lupicaris and C. pulicaris parous females. DL-Lactic acid significantly attracted more C. obsoletus/C. scoticus females and males, C. lupicaris females, C. pulicaris females and Culicoides punctatus females and males; whereas acetone increased only the captures of Culicoides achrayi.     

Preferred landing sites of Culicoides species (Diptera: Ceratopogonidae) on a horse in Israel and its relevance to summer seasonal recurrent dermatitis (sweet itch)

Six hundred and twenty culicoides of five species were collected from a bait horse at Kannot, Israel, between April and September 1986. Seventy-two per cent of the midges were collected from the belly and 27 per cent from the dorsal aspect of the body, ie, the sweet itch summer seasonal recurrent dermatitis (SSRD) zone. Midges were active mainly from half an hour prior to half an hour after sunset. Only Culicoides puncticollis, C imicola and C schultzei group were collected in considerable numbers. While C imicola was present continuously throughout the season, the appearance of the two other species was intermittent. Ninety-five per cent of C schultzei group and approximately 100 per cent of the C puncticollis prefer to land on and most probably bite the belly. Culicoides imicola showed a clear preference for the dorsal ridge which overlaps the (SSRD) itch zone, and 70 per cent of the midges were collected there, while 28 per cent were collected from the belly. A considerably high proportion of the midges of this species were parous, ie, part of the population lived long enough to bite more than once. Skin temperature measurements showed the belly to be the warmest part of the body. Wind speeds of two to three knots reduced the number of midges collected. The findings of this study, together with a previous one (Braverman et al 1983), incriminate C imicola as the likely principal agent of SSRD in Israel.     

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.     

Immunogenicity and allergenicity of Culicoides imicola (Diptera: Ceratopogonidae) extracts

Summer seasonal recurrent dermatitis (SSRD) or "sweet itch" is a seasonally occurring allergic dermatitis of horses provoked by biting midges. The allergic skin reactions have been attributed to allergens present in various Culicoides species. C. imicola is the suspected etiological agent of SSRD in Israel. Whole body extracts of this midge induced hypersensitivity reactions upon injection into susceptible horses and in this study attempts were made to define components of C. imicola which have immunogenic and allergenic properties. Immunogenic potency was evaluated by raising antisera to whole body extracts of C. imicola in rabbits and examining their reactivity towards fractionated extracts. Allergenic potency was examined by reacting fractionated extracts with horse sera. Humoral reactivity of susceptible and non susceptible horses was assayed by specific IgE and IgG ELISAs. Although there are many antigenic components in whole body extracts of C.imicola capable of eliciting an immune response, no conclusive evidence was obtained indicating that allergic reactivity was associated with increased IgE levels of defined specificity.     

Evidence for a new field Culicoides vector of African horse sickness in South Africa

Between February and May 1998, approximately 100 horses died of African horse sickness (AHS) in the cooler, mountainous, central region of South Africa. On 14 affected farms, 156,875 Culicoides of 27 species were captured. C. imicola Kieffer, hitherto considered the only field vector for AHS virus (AHSV), constituted <1% of the total Culicoides captured, and was not found on 29% of the farms. In contrast, 65% of the Culicoides were C. bolitinos Meiswinkel, and was found on all farms. Five isolations of AHSV were made from C. bolitinos, and none from 18 other species of Culicoides (including C. imicola).     

The effect of climate on the presence of Culicoides imicola in Italy

A model was developed to classify the Italian territories in relation to their suitability to harbour populations of Culicoides imicola and, as a consequence, also able to sustain a bluetongue (BT) epidemic. Italy was subdivided into 3507 10 x 10 km cells. In 546 cells at least one collection was made. The cell was considered the unit for all subsequent analyses. Culicoides were collected using Onderstepoort-type blacklight traps. Some traps were operated weekly at chosen sites; the remainder were moved almost daily to new sites. Only the results obtained during the peak August-November period were used, to exclude bias caused by the seasonality of C. imicola. Climate data for the period 1999-2001 were obtained from 80 weather stations. Multiple logistic regression was performed using the presence or absence of C. imicola in a specific cell as the dependent variable. Annual means of daily values for minimum temperature and minimum relative humidity, and the mean altitude above sea level, were the independent variables. The probability of occurrence of C. imicola in each grid cell was used to create a prediction map for Italy. The model was able to correctly classify 77.5% of the 546 grid cells in which at least one collection had been made. Culicoides imicola was found frequently through much of Sardinia, in parts of southern Italy, and further north along the Tyrrhenian coast, but was absent from along most of the Adriatic coast, and the internal mainland, and from most of Sicily. Six detailed maps are provided. Also mapped are areas where the probability of the occurrence of C. imicola is lower than 5%. This identification of possible mountainous C. imicola-free areas in central Italy could facilitate safer animal trade and transhumance, even if BT infections in traded animals or moving stock, were to go undetected. Needless to say this depends upon no cool-adapted species of Culicoides being involved in the transmission of BT disease.     

利用SYBR Green检测衣原体Real-Time PCR方法的建立

利用SYBR Green建立了检测种特异性衣原体的Real-Time PCR方法。本方法应用衣原体种特异性的高度保守特异引物，能够扩增627bp特异片段；使用定量标准基因组DNA，本方法能准确检测最少250fg衣原体DNA。Real-TimePCR方法与免疫荧光方法的检测结果表明：检测4种衣原体临床样本，Real-TimePCR敏感性均在96％～98%；特异性均为100％；这两种方法符合率达97％以上（n=60）；批内和批间重复性试验结果表明，本方法具有良好的准确性。本方法的建立对于快速、准确检测临床样本种特异性衣原体提供了一种切实有效的方法。     

Comparative descriptions of the pupae of five species of the Culicoides imicola complex (Diptera, Ceratopogonidae) from South Africa

The viruses causing the economically important livestock diseases of African horse sickness (AHS) and bluetongue (BT) are transmitted by biting midges of the genus Culicoides (Diptera, Ceratopogonidae). In the Old World the most important vectors of these diseases are Culicoides imicola Kieffer, 1913, Culicoides brevitarsis Kieffer, 1917 and Culicoides bolitinos Meiswinkel, 1989. All three of these vectors belong to the Imicola complex of the subgenus Avaritia Fox, 1955. This species complex now comprises 12 sibling species; ten occur in sub-Saharan Africa and are difficult to identify (based mostly on subtle variations in the wing latterns) and so additional methods of reliable identification are needed. The pupal exuviae of the five commonest sibling species (C. imicola, C. bolitinos, Culicoides loxodontis Meiswinkel, 1992, Culicoides tuttifrutti Meiswinkel, Cornet & Dyce, 2003 and Culicoides sp. # 107) harvested from a variety of large herbivore dung types and from decaying fruits, are described and illustrated in detail. It is shown that they can be differentiated clearly on a number of morphological characters and, furthermore, are separable into two distinct groups based (principally) on the shape of the respiratory organ. A key for identifying and differentiating these five pupae is provided. Also, the pupa of the Oriental-Australasian C. brevitarsis was compared with its allopatric sister taxon, C. bolitinos. Because they share a common larval habitat (cattle and buffalo dung) and are almost inseparable in the adult phenotype, the question of their possible synonymy is raised. However, their respective pupae could not be differentiated on gross morphology and so it is argued that this unresolved problem requires a molecular solution.     

Seasonal abundance and parity of Culicoides biting midges associated with livestock at Roma, Lesotho (Diptera: Ceratopogonidae)

A light-trap survey was undertaken of the species composition, seasonal abundance and parity of Culicoides at Roma, Lesotho, to establish whether the likely vectors for bluetongue and African horse sickness occur in this area as well as the chance of transmission. A total of 34 catches was made between 21 September 1985 and 24 September 1986; 32,819 Culicoides were caught belonging to 19 species. Culicoides numbers rapidly built up from December to a peak in February which implies that this may also be the optimum time for virus transmission. The number of Culicoides dropped sharply in April with the onset of cooler conditions. C. zuluensis was the dominant species forming 69.6% of the totalled catches, followed by C. pycnostictus with 11.7%. C. imicola, the only proven vector of bluetongue, was never abundant representing only 4.4% of the midges caught. The parous rate for each of the 2 commonest species was low, implying a low vector capacity.     

Afrotropical Culicoides: C (Avaritia) miombo sp. nov., a widespread species closely allied to C. (A.) imicola Kieffer, 1913 (Diptera: Ceratopogonidae)

Culicoides (Avaritia) miombo sp. nov. is described and illustrated from both sexes collected in northern Malawi. Two references in the literature have previously referred to this new species as either C. brosseti Vattier & Adam or C. imicola Kieffer. A further 4 references are discussed that most likely deal with C. miombo sp. nov. and not C. brosseti. C. miombo sp. nov. is apparently widespread in subtropical and tropical Africa and is now recorded from Zimbabwe, Botswana, South Africa, Nigeria and the Ivory Coast. There are also probable records from Angola, Burkina Faso, Zambia and eastern Madagascar. On the African mainland, both north and south of the equator, the pattern of distribution of C. miombo sp. nov. correlates strongly with that of drier Guineo-Congolian rainforest, and Sudanian and Zambezian woodlands, the latter known as miombo in southern Africa. These phytochoria and associated biota are sensitive to frost and experience relatively high temperatures and rainfall-3 factors that appear to limit the distribution of C. miombo sp. nov. to north of the 20-22 degrees C mean annual temperature isotherms in southern Africa. The new species is a member of the Imicola group which consists of 6 species confined to the Afrotropical (including Madagascar), Oriental and eastern Palaearctic regions. One species has in historic times spread to Australia. The worldwide distribution of each species is briefly discussed. It is suggested that the Imicola and Orientalis groups are separate lineages within the subgenus Avaritia. Culicoides miombo sp. nov. is compared with its closest African congeners C. imicola, C. pseudopallidipennis Clastrier and C. bolitinos Meiswinkel; 15 character states are used to separate C. miombo sp. nov. and C. imicola. The female antennal and palpal measurements of C. miombo sp. nov. are subjected to statistical analysis to highlight their taxonomic usefulness. The larval habitat of C. miombo sp. nov. is unknown.     

A method for extracting genomic DNA from individual elaphostrongyline (Nematoda: Protostrongylidae) larvae and differentiation of Elaphostrongylus spp. from Parelaphostrongylus spp. by PCR assay.

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.