RESUMO
Culicoides species of the Obsoletus group (Diptera: Ceratopogonidae) are potential vectors of bluetongue virus serotype 8 (BTV 8), which was introduced into central Western Europe in 2006. Correct morphological species identification of Obsoletus group females is especially difficult and molecular identification is the method of choice. In this study we present a new molecular tool based on probe hybridization using a DNA microarray format to identify Culicoides species of the Obsoletus group. The internal transcribed spacer 1 (ITS1) gene sequences of 55 Culicoides belonging to 13 different species were determined and used, together with 19 Culicoides ITS1 sequences sourced from GenBank, to design species-specific probes for the microarray test. This test was evaluated using the amplified ITS1 sequences of another 85 Culicoides specimens, belonging to 11 species. The microarray test successfully identified all samples (100%) of the Obsoletus group, identifying each specimen to species level within the group. This test has several advantages over existing polymerase chain reaction (PCR)-based molecular tools, including possible capability for parallel analysis of many species, high sensitivity and specificity, and low background signal noise. Hand-spotting of the microarray slide and the use of detection chemistry make this alternative technique affordable and feasible for any diagnostic laboratory with PCR facilities.
Assuntos
Ceratopogonidae/classificação , Ceratopogonidae/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Sequência de Bases , DNA Espaçador Ribossômico/genética , Feminino , Dados de Sequência Molecular , Filogenia , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
In response to the first bluetongue outbreak in Belgium a monitoring programme was started at the end of August 2006 to identify possible vectors transmitting the disease. Black light traps were deployed at 36 outbreak sites and captured 1959 Culicoides specimens belonging to 16 different species. Eighty four percent of the biting midges captured belonged to the C. obsoletus complex, among them C. obsoletus s.s., C. dewulfi and C. scoticus, three suspected bluetongue vectors. The Veterinary and Agrochemical Research Centre detected viral RNA in pools of individuals belonging to this complex. Culicoides pulicaris, a potential bluetongue vector in Italy, should yet not be excluded as a possible vector in Belgium as this species was frequently found around outbreak sites, notwithstanding this species is not easily captured with the trapping techniques used during this survey.
Assuntos
Vírus Bluetongue/crescimento & desenvolvimento , Ceratopogonidae/crescimento & desenvolvimento , Surtos de Doenças/veterinária , Insetos Vetores/virologia , Animais , Bélgica/epidemiologia , Bluetongue/epidemiologia , Bluetongue/transmissão , Bluetongue/virologia , Vírus Bluetongue/genética , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/transmissão , Doenças dos Bovinos/virologia , Ceratopogonidae/genética , Ceratopogonidae/virologia , Estudos Transversais , Insetos Vetores/genética , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , OvinosAssuntos
Alérgenos/imunologia , Basófilos/efeitos dos fármacos , Ceratopogonidae/química , Doenças dos Cavalos/diagnóstico , Hipersensibilidade/diagnóstico , Hipersensibilidade/imunologia , Mordeduras e Picadas de Insetos/imunologia , Animais , Basófilos/metabolismo , Histamina/metabolismo , Doenças dos Cavalos/imunologia , Cavalos , Especificidade da EspécieRESUMO
After the emergence of bluetongue (BT) in Belgium in 2006, two types of entomological surveys were initiated, the one to identify the local vector species, and the other to study their population dynamics. In the vector study, Culicoides were captured near farms with recently infected cattle or sheep; in the population study Culicoides were captured in two meadows situated in the BT-affected region. A total of 130 pools of parous, non-blood engorged female midges (with a mean of 7.5 midges per pool) were analysed with real-time reverse transcription PCR (RT-qPCR) targeting bluetongue virus (BTV) segment 5. To ensure the RNA integrity of the samples, all pools were also tested in a second RT-qPCR targeting Culicoides 18S rRNA, which served as an internal control. Seventeen pools with negative results for both 18S and BTV were excluded, most of which originated from the population survey. In the vector survey near outbreak sites, female midges of the obsoletus complex, including C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, dominated the black-light trap collections with 19 of 89 pools being BTV-positive. Moreover, all the collections from the vector survey included at least one positive pool of the obsoletus complex compared with only 20% collections (C. obsoletus/C. scoticus) in the population survey. The current study also revealed the presence of BTV RNA in one of five pools of C. pulicaris females captured near recent BT outbreaks, suggesting that this species might have played a role in transmission. Finally, the use of RT-qPCR for the recognition of new potential BTV vector species and the impact of an appropriate monitoring method and internal control are discussed.