A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep

We evaluated a combined microscopic-molecular approach for the diagnosis of key strongylid infections in sheep using panels of well-defined control and test samples. The method established is based on the separation of nematode eggs from faecal samples using a salt flotation procedure, the extraction and column-purification of genomic DNA, followed by real-time PCR and melting-curve analysis. Specific and semi-quantitative amplification from (a minimum of 0.1-2.0pg) genomic DNA of Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp., Cooperia oncophora, Oesophagostomum columbianum, Oesophagostomum venulosum or Chabertia ovina is achieved using a specific, forward oligonucleotide primer located in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) together with a conserved reverse primer in the large subunit of rDNA. Using a panel of well-defined genomic DNA samples from eggs from sheep monospecifically infected with H. contortus or Te. circumcincta, there was a correlation between cycle threshold (Ct) values in the PCR and numbers of egg per gram of faeces, thus allowing the semi-quantitation of parasite DNA in faeces. The findings of the present study indicate that a microscopic-molecular approach provides a useful tool for diagnosis, for epidemiological and ecological surveys as well as for integration into parasite monitoring, drug resistance (i.e. 'egg count reduction') testing or control programmes, particularly following semi- or full-automation.