Abstract
BackgroundTraditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. Recently, we developed an automated multiplexed-tandem (MT)-PCR platform for the diagnosis and identification patent infections with key genera/species of gastrointestinal nematodes of sheep and validated this approach in detailed experiments carried out in Australia. In the present study, we deployed this diagnostic platform in Scotland and Belgium to test samples from naturally infected sheep in these environments and to validate the MT-PCR platform relative to traditional diagnostic methods routinely used by local laboratories.ResultsMT-PCR detected all microscopy positive samples and there was a significant agreement between the results of the different test methods in terms of the species detected and their relative proportion in a test sample, however, for some samples, there were discrepancies between the results of the different test methods. Selective sequencing of purified MT-PCR products demonstrated the results to be 100% specific.ConclusionsThe MT-PCR platform is an advanced method for the species/genus-specific diagnosis of gastrointestinal nematode infections in small ruminants and has demonstrated utility when deployed in different countries and climatic zones. The platform is user-friendly due to the largely automated procedure and has high versatility in that it can achieve a specific diagnosis from different types of sample templates, including larval culture and faecal samples. With appropriate modifications of the primers used, the MT-PCR platform also provides potential for the diagnosis of a variety of other pathogens of veterinary or medical importance.
Highlights
Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity
There was a significant agreement between the results of multiplexed-tandem polymerase chain reaction (MT-PCR) and larval culture (LC) technique for the species detected as well as their determined proportions in an egg and/or LC sample (Table 1)
Scotland routinely works with small ruminant parasites and their identification whereas operators at Ghent, Belgium only infrequently deal with the larvae of sheep gastrointestinal nematodes and, have less experience with their morphological identification, which may have led to a higher incidence of misidentification
Summary
Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. In our previous studies conducted in Australia, this novel molecular diagnostic approach demonstrated to be highly sensitive and specific in the context of diagnosing naturally infected sheep It showed excellent agreement with the results of traditional diagnostic techniques such as FEC, LC and post-mortem total worm counts in regards to the presence/absence of different nematode species in naturally infected sheep [7]. Even though the sequences of the second internal transcribed spacer (ITS2), which are used during the molecular diagnostic process are highly conserved, some variation between and/or within different populations of parasites may occur, especially from different geographical locations [8, 9] In this proof-of-concept study, our aim was to deploy this new MT-PCR approach to the European environment and to determine its performance on samples obtained from sheep in Scotland and Belgium. We used samples from these countries to compare the results of this MT-PCR approach with the results of FEC and LC as performed by the relevant participating laboratories
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