Development Of Species-specific PCR Assay Research Articles

Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites

At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.

Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of important diseases affecting wild and domestic animals. During the last decade they have played a major role in the epidemiology of the largest bluetongue epizootic ever recorded in Europe, the disease is transmitted between hosts almost exclusively by bites of Culicoides midges and affects both domestic and wild ruminants however severe disease usually occurs in certain breeds of sheep and some species of deer. An accurate vector identification is of major importance in arthropod borne diseases surveillance, as great differences in vectorial capacity are found even between close species. Unfortunately, specialized taxonomic knowledge of Culicoides identification is rarely available in routine surveillance, mainly based on wing morphology. Recently, some European species of Culicoides belonging to the subgenus Avaritia Fox, 1955 and Culicoides Latreille, 1809 have been described as new bluetongue virus vectors. In the present study, by using a fragment of the barcode region (COI gene) we report the presence of up to 11 species within the subgenus Culicoides in Catalonia (NE Spain), a region recently affected by a bluetongue epizootic. The molecular analysis revealed new non-described cryptic species which were grouped in three complexes of morphologically similar species, two in the Pulicaris complex resembling Culicoides pulicaris, two in the Fagineus complex resembling Culicoides fagineus and three in the Newsteadi complex resembling Culicoides newsteadi. The phylogenetic relationships among them showed that cryptic species detected in both Pulicaris and Fagineus complexes were closely related, whereas those in the Newsteadi complex were more distant. Accurate analysis of all species using morphological and molecular approaches resulted in the detection of diagnostic metric traits for cryptic species and the design of several new species-specific single and multiplex PCR assays to identify unambiguously all the species, most of them still lacking a specific molecular diagnosis.