Real-time PCR detection for quantification of infection levels with Ostertagia ostertagi and Cooperia oncophora in cattle faeces.

A quantitative real-time polymerase chain reaction (qPCR) based on hydrolysis (TaqMan) probes is described for robust and sensitive detection of the infection levels with eggs and third stage larvae (L3) of Cooperia oncophora and Ostertagia ostertagi isolated from cattle faeces. The current microscopic method for identification of strongyle nematodes in cattle faeces is labour-intensive where reliable species determination also requires trained expertise, which is increasingly lacking. The goal of this study was to develop a sustainable non-labour intensive diagnostic qPCR assay to detect and determine the levels of infection with the two most common gastro-intestinal nematodes (GIN) in cattle faeces targeting the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) region (rDNA). According to our results, this procedure allows to reliably detect the relative proportions of eggs and L3 for each of the two species. This assay produced consistent results when mixtures with known numbers of L3 of both species were tested, although it was also demonstrated that the calculated copy numbers of ITS2 between single L3 sometimes varied very much. In addition, a positive correlation (r(2)=0.23) between the proportion of eggs and L3 in different paired samples collect in the field was observed for both species. Thus, for the first time a qPCR assay is reported, which can discriminate between the two most important cattle nematode parasites in temperate regions. This is of major importance to the livestock sector as it can be used with great precision to demonstrate strategic treatment efficacy that is important for the detection of anthelmintic resistance (AR).