Abstract
BackgroundThe diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.MethodsTwo multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC.ResultsMultiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.ConclusionsOur multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.Graphical
Highlights
The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques
These infections are responsible for parasitic gastroenteritis (PGE), which is a parasitic disease of major socioeconomic importance in farming communities worldwide [2]
The only exception was found in the detection system for Nematodirus where 1 ng of T. circumcincta DNA was non- amplified with Quantification cycle (Cq) 34.73 (Table 1)
Summary
The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Grazing ruminants are infected with several species of GINs whose unique mix contributes to the sum of the clinical effects and determines the severity of infections In combination, these infections are responsible for parasitic gastroenteritis (PGE), which is a parasitic disease of major socioeconomic importance in farming communities worldwide [2]. Members of the genera Nematodirus and Cooperia and those in the superfamily Strongyloidea (genera Chabertia and Oesophagostomum) commonly parasitize sheep and goats These latter species cause disease only in exceptional circumstances but may contribute to PGE. Even regular observation of animals may not reveal the extent of parasite burden until PGE develops into a severe clinical form [2]
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