An automated, multiplex-tandem PCR platform for the diagnosis of gastrointestinal nematode infections in cattle: An Australian-European validation study

Detecting the genera and species of gastrointestinal (GI) nematode infections in faecal samples obtained from cattle requires the incubation of faeces ('larval culture') followed by identification of the third-stage larvae that are harvested after 10-14 days. Substantial research in the development of PCR-based methods for the rapid and specific identification GI nematodes has been conducted for small ruminants, whilst only few such assays have been developed for cattle. In the present paper we describe the development of an automated, robotic PCR platform for the detection and genus and/or species-specific identification of GI nematodes from bovine faecal samples. This test was then validated using samples from different regions of three countries (Australia, Belgium and Scotland). The PCR platform was found to be highly sensitive and specific for the identification of the important GI nematodes in naturally infected cattle (both estimates > 90%). The PCR platform can also estimate the percentage of genera or species present in a mixed-species infection, and was found superior to larval culture in terms of speed (1-2 days versus 1-2 weeks for culture), sensitivity and specificity. The PCR was simple to use and the operator requires no knowledge or experience to identify the nematodes present, compared to larval culture where even experienced operators can make substantial errors due to considerable overlap in the published characteristics of key species.