BACKGROUND: 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. 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. METHODS: 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. 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. RESULTS: Multiplex 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. CONCLUSIONS: 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. 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.

• Keywords
• Cell-free DNA, Gastrointestinal nematode, Multiplex detection, Real-time PCR, Sheep,
• MeSH
• Feces parasitology   MeSH
• Gastrointestinal Diseases diagnosis   parasitology   veterinary   MeSH
• Gastrointestinal Tract parasitology   MeSH
• Nematoda classification   genetics   MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Nematode Infections diagnosis   parasitology   veterinary   MeSH
• Sheep Diseases diagnosis   parasitology   MeSH
• Sheep MeSH
• Parasite Egg Count MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.

• Keywords
• MOL-PCR, bioterrorism, biothreat bacteria, detection panel, magnetic bead,
• Publication type
• Journal Article MeSH