The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

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

Author Correction: A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay

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A deep exploration of the transcriptome and "excretory/secretory" proteome of adult Fascioloides magna

Parasitic liver flukes of the family Fasciolidae are responsible for major socioeconomic losses worldwide. However, at present, knowledge of the fundamental molecular biology of these organisms is scant. Here, we characterize, for the first time, the transcriptome and secreted proteome of the adult stage of the "giant liver fluke," Fascioloides magna, using Illumina sequencing technology and one-dimensional SDS-PAGE and OFFGEL protein electrophoresis, respectively. A total of ~54,000,000 reads were generated and assembled into ~39,000 contiguous sequences (contigs); ~20,000 peptides were predicted and classified based on homology searches, protein motifs, gene ontology, and biological pathway mapping. From the predicted proteome, 48.1% of proteins could be assigned to 384 biological pathway terms, including "spliceosome," "RNA transport," and "endocytosis." Putative proteins involved in amino acid degradation were most abundant. Of the 835 secreted proteins predicted from the transcriptome of F. magna, 80 were identified in the excretory/secretory products from this parasite. Highly represented were antioxidant proteins, followed by peptidases (particularly cathepsins) and proteins involved in carbohydrate metabolism. The integration of transcriptomic and proteomic datasets generated herein sets the scene for future studies aimed at exploring the potential role(s) that molecules might play at the host–parasite interface and for establishing novel strategies for the treatment or control of parasitic fluke infections

Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses

Abstract Background Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. Methods The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood–saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. Results The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood–saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030–0.9998 for SNVs and between 0.8883–0.9991 for small-indels in the case of the WGS protocol, and between 0.8643–0.999 for SNVs and between 0.7781–1.000 for small-indels in the case of the WES protocol. Conclusion Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used

Paragonimiasis

[Extract] Paragonimiasis is a zoonotic disease caused by lung flukes of the genus Paragonimus. Humans usually become infected by eating freshwater crabs or crayfish containing encysted metacercariae of these worms. However, an alternative route of infection exists: ingestion of raw meat from a mammalian paratenic host. Adult worms normally occur in pairs in cysts in the lungs from which they void their eggs via air passages. The pulmonary form is typical in cases of human infection due to P. westermani, P. heterotremus, and a few other species (Table 5.1). Worms may occupy other sites in the body, notably the brain, but lung flukes have made their presence felt in almost every organ. Ectopic paragonimiasis is particularly common when infection is due to members of the P. skrjabini complex (Table 5.1). Human paragonimiasis occurs primarily in the tropics and subtropics of Asia, Africa, and the Americas, with different species being responsible in different areas (Table 5.1)