RESUMO
A PCR-based assay for identification of six species of Pratylenchus common in California is described. In this assay, five forward species-specific primers were designed from the internal variable portion of the D3 expansion region of the 26S rDNA and were each used with a single, common reverse primer. The optimized species-specific primers produced unique amplicons from their respective target and did not amplify DNA from other Pratylenchus species. With this assay we were able to identify single females to species level. This method obviates the need for subsequent RFLP or sequence analysis of the PCR product and can be used as a rapid diagnostic tool in epidemiological and management studies.
RESUMO
Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.
Assuntos
Metastrongyloidea/classificação , Focas Verdadeiras/parasitologia , Infecções por Strongylida/veterinária , Animais , DNA de Helmintos/análise , DNA de Helmintos/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Feminino , Masculino , Metastrongyloidea/anatomia & histologia , Metastrongyloidea/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico/genética , Análise de Sequência de DNA , Infecções por Strongylida/parasitologiaRESUMO
We used nucleotide sequences of the large subunit ribosomal genes (26S rDNA) to examine evolutionary relationships among species of the genus Pratylenchus (Order: Tylenchida, Family: Pratylenchidae), commonly known as root-lesion nematodes. Ten species of Pratylenchus were studied including, P. penetrans, P. crenatus, P. minyus, P. vulnus, P. thornei, P. musicola, P. coffeae, P. hexincisus, P. scribneri, and P. brachyurus. The species Hirschmanniella belli, Meloidogyne javanica, Heterorhabditis bacteriophora, Nacobbus aberrans, Radopholus similis, and Xiphinema index were used as outgroups. Based on parsimony analyses of approximately 307 aligned nucleotides of the D3 expansion region of the 26S rDNA, it is clear that species of Pratylenchus are a paraphyletic assemblage. The outgroup taxon H. belli shares a common ancestor with the clade that includes P. vulnus and P. crenatus while N. aberrans and R. similis share a common ancestor with 5 other species included in this study.
Assuntos
DNA Ribossômico/genética , Nematoides/genética , Filogenia , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Variação Genética , Modelos Genéticos , Dados de Sequência Molecular , Nematoides/classificação , Reação em Cadeia da Polimerase , RNA Ribossômico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Tylenchoidea/genéticaRESUMO
Individuals of a new species of trichostrongyloid nematode of the genus Vexillata were collected from the intestines of the coarse-haired pocket mouse Chaetodipus hispidus from eastern New Mexico. This new species possesses general features of the genus Vexillata but may be recognized as distinct from all other species by unique features of the dorsal ray of the caudal bursa and the synlophe.
