RESUMO
Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.
Assuntos
Variação Genética , Doenças das Cabras/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Nematodirus/classificação , Nematodirus/genética , Doenças dos Ovinos/parasitologia , Infecções por Strongylida/veterinária , Animais , China , Análise por Conglomerados , Primers do DNA/genética , DNA de Helmintos/química , DNA de Helmintos/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Cabras , Dados de Sequência Molecular , Nematodirus/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade , Análise de Sequência de DNA , Ovinos , Infecções por Strongylida/parasitologiaRESUMO
AIM: To compare the prevalence of Nematodirus spathiger and N. filicollis on a sample of farms in the lower South Island with the lower and central North Island of New Zealand by way of a presence/absence survey. METHODS: A presence/absence non-random survey was conducted in which pooled faeces from lambs from 50 farms in the lower and central North Island (n=22) and lower South Island (n=28) were cultured, with and without a post-culture period of chilling, to produce third-stage parasitic nematode larvae (L3). After recovery using the Baermann technique, individual L3 were identified to Nematodirus spp. using a PCR assay. This identified the species that were present on each farm that were then used to compare the regions. RESULTS: Nematodirus filicollis was present in 38/50 (76%) samples from which Nematodirus spp. larvae were cultured, compared with 50/50 (100%) in the case of N. spathiger. No regional differences were observed in the prevalence of either species (p=0.74). Of the Nematodirus spp. L3 recovered from the unchilled samples, 415/428 (97%) were N. spathiger and 13/428 (3%) N. filicollis. After chilling 594/695 (85%) of the Nematodirus spp. L3 were N. spathiger and 101/695 (15%) were N. filicollis. CONCLUSION: Despite the non-random nature of the study, the evidence suggests that both these species are likely to occur sympatrically on most sheep farms throughout New Zealand. In general N. filicollis eggs did not hatch without a period of chilling and this has implications for the identification of these larvae using conventional culture methods.
