To a lesser extent, members of the genera Nematodirus and Cooperia and those in the superfamily Strongyloidea (genera Chabertia and Oesophagostomum) also commonly parasitize sheep and goats. In small ruminants, the GINs of greatest importance belong to the superfamily Trichostrongyloidea, and the most important genera are Haemonchus, Teladorsagia, and Trichostrongylus. In combination, these infections are responsible for parasitic gastroenteritis (PGE), which is a parasitic disease of major socioeconomic importance in farming communities worldwide. Typically, 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. The higher the worm load, the greater the impact on animal health, productivity, and welfare.
Graphical AbstractĪll grazing livestock are exposed to gastrointestinal nematode (GIN) infections. 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.
Our 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. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. 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. Multiplex real-time PCR assays showed great specificity to target nematodes. 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.
BIO RAD CFX MANAGER CQ VALUES SERIES
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. 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. Two 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. 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. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. 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.