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Increased levels of guinea worm (GW) disease transmission among dogs in villages along the Chari River in Chad threaten the gains made by the GW Eradication Program. Infected dogs with preemergent worm blisters are difficult to proactively identify. If these dogs are not contained, blisters can burst upon submersion in water, leading to the contamination of the water supply with L1 larvae. Guinea worm antigens previously identified using sera from human dracunculiasis patients were coupled to polystyrene beads for multiplex bead assay analysis of 41 non-endemic (presumed negative) dog sera and 39 sera from GW-positive dogs from Chad. Because commercially available anti-dog IgG secondary antibodies did not perform well in the multiplex assay, dog IgGs were partially purified, and a new anti-dog IgG monoclonal antibody was developed. Using the new 4E3D9 monoclonal secondary antibody, the thioredoxin-like protein 1-glutathione-S-transferase (GST), heat shock protein (HSP1)-GST, and HSP2-GST antigen multiplex assays had sensitivities of 69-74% and specificities of 73-83%. The domain of unknown function protein 148 (DUF148)-GST antigen multiplex assay had a sensitivity of 89.7% and a specificity of 85.4%. When testing samples collected within 1 year of GW emergence (n = 20), the DUF148-GST assay had a sensitivity of 90.0% and a specificity of 97.6% with a receiver-operating characteristic area under the curve of 0.94. Using sera from two experimentally infected dogs, antibodies to GW antigens were detected within 6 months of exposure. Our results suggest that, when used to analyze paired, longitudinal samples collected 1-2 months apart, the DUF148/GST multiplex assay could identify infected dogs 4-8 months before GW emergence.

DNA damage causes cancer, impairs development and accelerates aging. Transcription-blocking lesions and transcription-coupled repair defects lead to developmental failure and premature aging in humans. Following DNA repair, homeostatic processes need to be reestablished to ensure development and maintain tissue functionality. Here, we report that, in Caenorhabditis elegans, removal of the WRAD complex of the MLL/COMPASS H3K4 methyltransferase exacerbates developmental growth retardation and accelerates aging, while depletion of the H3K4 demethylases SPR-5 and AMX-1 promotes developmental growth and extends lifespan amid ultraviolet-induced damage. We demonstrate that DNA-damage-induced H3K4me2 is associated with the activation of genes regulating RNA transport, splicing, ribosome biogenesis and protein homeostasis and regulates the recovery of protein biosynthesis that ensures survival following genotoxic stress. Our study uncovers a role for H3K4me2 in coordinating the recovery of protein biosynthesis and homeostasis required for developmental growth and longevity after DNA damage.

The Proterodiplostomidae Dubois, 1936 is a relatively small family of diplostomoidean digeneans parasitising the intestines of reptilian hosts associated with freshwater environments in tropical and subtropical regions. The greatest diversity of proterodiplostomids is found in crocodilians, although some parasitise snakes and turtles. According to the most recent revision, the Proterodiplostomidae included 17 genera within 5 subfamilies. Despite the complex taxonomic structure of the family, availability of testable morphology-based phylogenetic hypotheses and ancient hosts, molecular phylogenetic analyses of the group were practically lacking. Herein, we use novel DNA sequence data of the nuclear lsrRNA gene and mitochondrial cox1 gene from a broad range of proterodiplostomid taxa obtained from crocodilian, fish, and snake hosts on four continents to test the monophyly of the family and evaluate the present morphology-based classification system of the Proterodiplostomidae in comparison with the molecular phylogeny. This first detailed phylogeny for the Proterodiplostomidae challenges the current systematic framework. Combination of molecular phylogenetic data with examination of freshly collected quality specimens and re-evaluation of morphological criteria resulted in a number of systematic and nomenclatural changes along with a new phylogeny-based classification of the Proterodiplostomidae. As the result of our molecular and morphological analyses: (i) the current subfamily structure of the Proterodiplostomidae is abolished; (ii) three new genera, Paraproterodiplostomum n. g., Neocrocodilicola n. g. and Proteroduboisia n. g., are described and Pseudoneodiplostomoides Yamaguti, 1954 is restored and elevated from subgenus to genus level; (iii) two new species, Paraproterodiplostomum currani n. g., n. sp. and Archaeodiplostomum overstreeti n. sp., are described from the American alligator in Mississippi, USA. Comparison of the structure of terminal ducts of the reproductive system in all proterodiplostomid genera did not support the use of these structures for differentiation among subfamilies (or major clades) within the family, although they proved to be useful for distinguishing among genera and species. Our study includes the first report of proterodiplostomids from Australia and the first evidence of a snake acting as a paratenic host for a proterodiplostomid. A key to proterodiplostomid genera is provided. Questions of proterodiplostomid-host associations parasitic in crocodilians are discussed in connection with their historical biogeography. Our molecular phylogeny of the Proterodiplostomidae closely matches the current molecular phylogeny of crocodilians. Directions for future studies of the Proterodiplostomidae are outlined. Electronic supplementary material The online version of this article (10.1007/s11230-020-09928-2) contains supplementary material, which is available to authorized users.

Prior to the present study, species of the trematode family Monorchiidae Odhner, 1911 had been reported from four of the ten families of tetraodontiform fishes: the Balistidae, Monacanthidae, Ostraciidae and Tetraodontidae. Here we report the first monorchiid from the family Triacanthidae, Pseudohurleytrema yolandae n. sp. infecting Tripodichthys angustifrons (Hollard), from Moreton Bay, Queensland, Australia. The species conforms well to the morphological concept of the subfamily Hurleytrematinae Yamaguti, 1958, and the genus Pseudohurleytrema Yamaguti, 1954, in the possession of filamented eggs, a bipartite terminal organ, and a unipartite seminal vesicle. Relative to the other three recognised species of Pseudohurleytrema, the present species is distinctive in the size of the testis and eggs, position of the ovary, and the form of the vitellarium and excretory vesicle. We consider Pseudohurleytrema magnum Kaikabad & Bilqees in Bilqees, 1991 as a species inquirenda. Sequence data for the 28S ribosomal RNA gene and cox1 mitochondrial gene were generated for P. yolandae, providing the first molecular data for the genus. Phylogenetic analysis showed that P. yolandae does not form a clade with the other three hurleytrematine genera for which there are molecular data (Helicometroides Yamaguti, 1934, Hurleytrematoides Yamaguti, 1953 and Provitellus Dove & Cribb, 1998), forming a poorly-supported clade with Proctotrema addisoni Searle, Cutmore & Cribb, 2014 within the clade of the subfamily Monorchiinae Odhner, 1911. The four hurleytrematine genera resolved as four distinct clades, indicating that the current subfamilial classification requires comprehensive revision.

Schistosomiasis is a globally important helminthic disease of humans and animals, and it is the second most common parasitic disease after malaria. Eggs produced by mature females are responsible for the disease’s occurrence and spread. Frzb2, a secreted frizzled-related protein, can inhibit Wnt signalling by competitive binding to the specific frizzled protein receptor. In this study, the complete gene sequence of SjFrzb2 was obtained by using 3′-rapid amplification of cDNA ends technology. SjFrzb2 transcript levels at different stages of S. japonicum maturation were evaluated by quantitative real-time RT-PCR analysis. SjFrzb2 was expressed at all developmental stages examined and exhibited the highest transcription level in 7-day-old worms, then gradually decreased during the growth and developmental stages to reach the lowest level at 18 days post-infection. SjFrzb2 gene expression was higher in female worms than in male worms and was significantly higher in female worms from a single-sex infection than in female worms from a bisexual infection. The functions of SjFrzb2 were explored via a small interfering RNA-based gene silencing approach and the soaking method. The results showed that SjFrzb2 gene knockdown impaired the growth and development of S. japonicum in mice, affecting not only the survival and morphological structure of the worms but also their reproductive ability and the viability of the produced eggs. Collectively, these observations imply that Frzb2 may be a novel target for the development of immuno- and/or small molecule-based therapeutics to control schistosomiasis fecundity and transmission.

Human sparganosis is a cestode infection which is neglected as a differential diagnosis outside endemic countries. Diagnosis and therapy may be challenging depending on the clinical presentation and anatomic localization. The disease manifests predominantly as subcutaneous nodule(s) or intracranial mass lesion(s). Infection is primarily acquired by ingesting raw or undercooked amphibian or reptile flesh or by drinking water containing copepods. We report an unusual case of subcutaneous Spirometra erinaceieuropaei sparganosis presenting with two nonmigratory nodules in close proximity to each other on the right thigh of a Thai woman living in Switzerland.

Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases.

Taenia martis is a tapeworm dwelling in the intestine of mustelids and a rare zoonotic cysticercosis pathogen in its larval stage. The metacestode is morphologically very similar to more prevalent cysticercosis parasites, such as the larvae of Taenia solium and Taenia crassiceps, and may be indistinguishable from other metacestodes on histological sections. However, the epidemiology of human T. martis infections is different, and for prognosis, prevention, and detection of natural parasite reservoirs, the species should be identified. We here report the molecular identification of a T. martis larva located in the pouch of Douglas in a female German patient who underwent surgery for endometriosis. This case represents the fifth human infection described worldwide; all previous cases were also in European women, involving the eye, brain, and the peritoneum.

This study employed single nucleotide polymorphisms (SNPs) to determine the genetic variability present in 26 isolates of Rotylenchulus reniformis from Louisiana, Mississippi, Arkansas, South Carolina, Georgia, Hawaii, and Alabama. Genomic DNA from reniform nematode was extracted and increased quantitatively using the process of whole genome amplification. More than 162 putative SNPs were identified, 31 of which were tested using a KASP kompetitive allele-specific PCR genotyping assay. Of the SNPs tested, 13, 17, and 19 SNPs revealed genetic variability within reniform nematode isolates from Louisiana, Mississippi, and Arkansas, respectively. Seven SNPs elucidated genetic differences among isolates of reniform nematode from Louisiana, Mississippi, and Arkansas. Eight SNPs determined genetic variability among individual isolates from South Carolina, Georgia, Hawaii, and Alabama. This study is the first to report genetic variability in geographic isolates of reniform nematode employing a SNP assay. This study also demonstrated that SNP markers can be used to evaluate isolates of R. reniformis and could be useful to assess their genetic diversity, origin, and distribution. Such information would be extremely useful in resistance breeding programs.

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode affecting over 500 plant species worldwide. Since 2012, garlic producers from Ontario and Quebec have been particularly affected with economic losses caused by this pest. Reproduction of D. dipsaci on a particular host depends on its biological race, and races are unknown for these populations from eastern Canada. As a polyphagous pest, D. dipsaci can possibly be a threat and have negative impact on many crops grown in Quebec, such as field and vegetable crops (e.g., onion). In this study, the host range of four populations of D. dipsaci from Quebec and Ontario was determined in a greenhouse experiment using 11 crops. Garlic, onion, and green onion showed high susceptibility to the nematode, whereas reproduction on potato was poor. No reproduction was observed on corn, soybean, barley, alfalfa, mustard, carrot, and lettuce. These crops could therefore be used as rotational crops in a control program. Thirty-two populations of D. dipsaci were also genetically characterized using genotyping-by-sequencing. The comparison of allele frequencies at 481 loci showed that most of the populations had a genotype similar to a reference population from northern Ontario. However, a sample from eastern Quebec exhibited a distinct genotype and will require further phenotyping in a greenhouse to preclude the possibility of a different race.

Taeniasis is a cosmopolitan helminthic disease caused by Taenia species, which included Taenia solium, Taenia saginata, and Taenia asiatica. These parasites typically infect the small intestine, but cases of aberrant migration have been reported. We treated a 70-year-old man who presented with vomiting and colicky abdominal pain. On physical examination, Murphy’s sign was positive, and laboratory findings indicated severe inflammation. Computed tomography and magnetic resonance cholangiopancreatography revealed typical features of cholecystitis. An 82-cm-long, slender and degenerated, parasite-like organism was aspirated through a percutaneous transhepatic gallbladder drainage tube. After extensive washing of the organism, we detected yellowish-brown colored, spherical 37.9 × 33.8-µm-sized taenid eggs with thick transverse striations. Hematoxylin–eosin-stained worm sections also contained Taeniidae eggs. Polymerase chain reaction amplification of DNA extracted from the worm with species-specific cytochrome c1 (cox1) primer sets detected a T. solium–specific fragment. Because of sustained high fever combined with inflammatory signs, the patient underwent laparoscopic cholecystectomy and inflamed gallbladder removal. A histopathologic specimen demonstrated chronic reactive cholecystitis. The patient’s fever and leukocytosis rapidly resolved after surgery. We experienced an uncommon case of biliary taeniasis representing cholecystitis caused by adult worm of T. solium.

Cysticercosis is a cosmopolitan zoonotic parasitic disease infected by larval ofTaenia solium(T. solium). Several drugs for the treatment of cysticercosis, such as praziquantel, albendazole, and mebendazole, have certain toxicity and side effects. Considering that there is no vaccine available, we studied a new vaccine for cysticercosis in this study. The completeTSOL18gene and the optimizedSP-TSOL18gene fragments were obtained using PCR-based accurate synthesis method. The secretory and intracellular recombinant pMG36e-SP-TSOL18/Lactococcus lactis(L. lactis) and pMG36e-TSOL18/L. lactisvaccines ofT. soliumwere prepared. Immune responses in mice orally immunized with these two recombinantL. lactisvaccines were analyzed by the determination of specific antibodies (IgG, IgG1, IgG2a, and sIgA) in serum, spleen lymphocyte proliferation, and cytokines (IL-2, IFN-γ, IL-4, and IL-10) in spleen lymphocyte culture supernatant. Our results showed that, after the first immunization, in these two recombinantL. lactisvaccine groups, the levels of serum specific IgG, IgG2a, and IgG1 increased on 14–56 d and reached the highest level on days 42, 42, and 28, respectively. The level of specific sIgA of intestinal mucosa also increased on 14–56 d and reached the highest level on day 42. The level of spleen lymphocyte proliferation increased on 14–56 d and reached the highest level on day 42. The levels of IL-2, IFN-γ, IL-4, and IL-10 in spleen lymphocyte culture supernatant increased on 14–56 d and reached the highest level on days 42, 42, 28, and 28, respectively. These results indicated that the recombinant pMG36e-SP-TSOL18/L. lactisand pMG36e-TSOL18/L. lactisvaccines can induce specific cellular, humoral, and mucosal immune responses in mice with oral vaccination. More importantly, the recombinant pMG36e-SP-TSOL18/L. lactisvaccine has a better immune effect. In summary, these results demonstrated the possibility of usingL. lactisstrain as a vector to deliver protective antigens ofT. solium.

Filarial parasitic nematodes (Filarioidea) cause substantial disease burden to humans and animals around the world. Recently there has been a coordinated global effort to generate, annotate, and curate genomic data from nematode species of medical and veterinary importance. This has resulted in two chromosome-level assemblies (Brugia malayi and Onchocerca volvulus) and 11 additional draft genomes from Filarioidea. These reference assemblies facilitate comparative genomics to explore basic helminth biology and prioritize new drug and vaccine targets. While the continual improvement of genome contiguity and completeness advances these goals, experimental functional annotation of genes is often hindered by poor gene models. Short-read RNA sequencing data and expressed sequence tags, in cooperation with ab initio prediction algorithms, are employed for gene prediction, but these can result in missing clade-specific genes, fragmented models, imperfect mapping of gene ends, and lack of isoform resolution. Long-read RNA sequencing can overcome these drawbacks and greatly improve gene model quality. Here, we present Iso-Seq data for B. malayi and Dirofilaria immitis, etiological agents of lymphatic filariasis and canine heartworm disease, respectively. These data cover approximately half of the known coding genomes and substantially improve gene models by extending untranslated regions, cataloging novel splice junctions from novel isoforms, and correcting mispredicted junctions. Furthermore, we validated computationally predicted operons, manually curated new operons, and merged fragmented gene models. We carried out analyses of poly(A) tails in both species, leading to the identification of non-canonical poly(A) signals. Finally, we prioritized and assessed known and putative anthelmintic targets, correcting or validating gene models for molecular cloning and target-based anthelmintic screening efforts. Overall, these data significantly improve the catalog of gene models for two important parasites, and they demonstrate how long-read RNA sequencing should be prioritized for ongoing improvement of parasitic nematode genome assemblies., Author summary Filarial parasitic nematodes are vector-borne parasites that infect humans and animals. Brugia malayi and Dirofilaria immitis are transmitted by mosquitoes and cause human lymphatic filariasis and canine heartworm disease, respectively. Recent years have seen a dramatic increase in genomic and transcriptomic data sets and the concomitant increase in innovative strategies for drug target identification, validation, and screening. However, while the completeness of genome assemblies of filarial parasitic nematodes has seen steady improvements, the reliability of gene models has not kept pace, hindering cloning efforts. Long-read RNA sequencing technologies are uniquely able to improve gene models, but have not been widely used for the causative agents of neglected tropical diseases. Here, we report the improvement of gene models in both B. malayi and D. immitis by long-read RNA sequencing. We identified novel operons, deprecated false positive operons, identified dozens of novel genes, and described the parameters of polyadenylation. We also focused on putative anthelmintic targets, identifying novel isoforms and correcting gene models. These data substantially increase the trustworthiness of gene models in these two species and demonstrate how long-read sequencing approaches should be prioritized in the continued improvement of genome assemblies and their gene annotations.

There is an urgent need for the development of new, improved vaccine adjuvants against T. spiralis infection. Polysaccharides are effective, safe, and biodegradable as adjuvant. In our study, we first observed the protective efficacy of lentinan as adjuvant against helminth T. spiralis infection. Recombinant T. spiralis Serpin (rTs-Serpin) immunoscreened from a cDNA library of T. spiralis, as a vaccine, protect host against Trichinella infection. The reduction rate of helminth burden of rTs-Serpin+lentinan-immunized mice was significantly increased compared with rTs-Serpin+FCA -immunized mice. rTs-Serpin+lentinan induced IgG1-dominant immune response and higher levels of IFN-γ and IL-4. rTs-Serpin+lentinan displayed a lower reduction rate of parasite burden in NLRP3-/- mice than that in WT mice and lower level of IgG1 than that in WT mice. The level of IL-4, but not IFN-γ, from NLRP3-/- mice immunized by rTs-Serpin+lentinan was significantly lower than that from WT mice, suggesting that NLRP3 is associated with rTs-Serpin+lentinan -triggering Th2 protective immunity against T. spiralis infection. In summary, we revealed that lentinan was a novel adjuvant against T. spiralis infection via NLRP3. NLRP3 therefore represents an important target for adjuvant discovery and the control of T. spiralis infection.

Among gastrointestinal nematodes, haematophagous strongylids Haemonchus contortus and Ashworthius sidemi belong to the most pathogenic parasites of both domestic and wild ruminants. Correct identification of parasitic taxa is of crucial importance in many areas of parasite research, including monitoring of occurrence, epidemiological studies, or testing of effectiveness of therapy. In this study, we identified H. contortus and A. sidemi in a broad range of ruminant hosts that occur in the Czech Republic using morphological/morphometric and molecular approaches. As an advanced molecular method, we employed qPCR followed by High Resolution Melting analysis, specifically targeting the internal transcribed spacer 1 (ITS-1) sequence to distinguish the two nematode species. We demonstrate that High Resolution Melting curves allow for taxonomic affiliation, making it a convenient, rapid, and reliable identification tool.

Specimens of Oncicola venezuelensis (Marteau, 1977) were recovered from fragments of intestinal tissue of a female Puma concolar (Linn, 1771) found dead in Petrópolis, Rio de Janeiro in 2017. A total of 140 helminths were recovered. Five males and 5 females of the helminths were analyzed morphologically as well as 50 parasite eggs recovered in intestinal contents. Morphologically, these helminths were compatible with the genus Oncicola, because of the size and shape of the proboscis, the size and disposition of the lemnisci and the morphometry of the eggs, in which the external membrane of the shell was delicate and clear. From histopathology, the helminths were deeply embeded in the mucosa reaching up to the muscle layer. One specimen was also identified molecularly with universal primers that amplified the eukaryote region ITS1-5.8S-ITS2. The helminth showed 99% identity with the gene sequence of O. venezuelensis deposited in GenBank. It is important to emphasize, this parasite has been very little reported in the literature, which reinforces the importance of this report. Resumo Espécimes de Oncicola venezuelensis (Marteau, 1997) foram recuperados de fragmentos do tecido intestinal de uma fêmea de Puma concolor (Linn, 1771) encontrada morta em Petrópolis, Rio de Janeiro, em 2017. Um total de 140 helmintos foram recuperados. Cinco machos e 5 cinco fêmeas dos helmintos foram analisados morfologicamente, bem como 50 ovos dos parasitos recuperados no conteúdo intestinal. Morfologicamente, esses helmintos eram compatíveis com o gênero Oncicola, devido ao tamanho e formato da probóscide, o tamanho e disposição do leminisco e a morfometria dos ovos, que apresentaram membrana externa da casca delicada e clara. A partir da histopatologia, pode-se verificar que os helmintos estavam profundamente inseridos na mucosa, atingindo até a camada muscular. Um espécime também foi identificado molecularmente com primers universais que amplificam a região ITS-1.5.8S.ITS-2. Após as análises moleculares, foi verificado que os helmintos apresentavam 99% de identidade com sequência gênica de O. venezuelensis que está depositada no Genbank. É importante enfatizar, que esse parasito foi muito pouco relatado na literatura, demonstrando a importância deste relato.

Background Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. Methodology/Principal findings We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34–117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34–117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34–117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34–117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to inflammatory responses. Conclusions/Significance These results offer new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, and places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities., Author summary Sm16 is a low molecular weight protein (~16kDa) secreted by Schistosoma mansoni, a causative agent of human schistosomiasis. The molecule is secreted by the infectious cercariae during skin invasion and performs an immune-suppressive function to protect the invading parasite from immune attack. Using phylogenetic and gene structure analysis we show that Sm16 homologues of parasites belonging to the Schistosomatoidea superfamily of digenean worms are members of the helminth defence molecule (HDM) family that are potent immune-modulators and exclusive to trematode species. Structural analyses revealed that Sm16, much like other HDMs, consists predominantly of an amphipathic alpha-helix. Sm16 is highly expressed in the cercariae and eggs of S. mansoni but not male or female adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. A synthetic form of the molecule, termed Sm16 (34–117), was shown to bind to and enter immune cells (macrophages) and induce a weak pro-inflammatory response. However, this peptide also blocked the pro-inflammatory effects of bacterial endotoxin (lipopolysaccharide, LPS). Analysis of the transcriptome of Sm16 (34–117)-stimulated macrophages in the presence or absence of LPS suggests that it mediates immunomodulatory activity via signalling pathways that are intricately involved in regulating cellular metabolism (fatty acid, cholesterol and glucose homeostasis) and central to inflammatory responses. These new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities.

Filarial nematodes can cause debilitating diseases in humans. They have complicated life cycles involving an insect vector and mammalian hosts, and they go through a number of developmental molts. While whole genome sequences of parasitic worms are now available, very little is known about transcription factor (TF) binding sites and their cognate transcription factors that play a role in regulating development. To address this gap, we developed a novel motif prediction pipeline, Emotif Alpha, that integrates ten different motif discovery algorithms, multiple statistical tests, and a comparative analysis of conserved elements between the filarial worms Brugia malayi and Onchocerca volvulus, and the free-living nematode Caenorhabditis elegans. We identified stage-specific TF binding motifs in B. malayi, with a particular focus on those potentially involved in the L3-L4 molt, a stage important for the establishment of infection in the mammalian host. Using an in vitro molting system, we tested and validated three of these motifs demonstrating the accuracy of the motif prediction pipeline., Author summary Diseases caused by parasitic worms such as the filariae are among the leading causes of morbidity in the developing world. Very little is known about how development is regulated in these vector-transmitted parasites. We have developed a computational method to identify motifs that correspond to transcription factor binding sites in the genome of the parasitic worm, Brugia malayi, one of the causative agents of lymphatic filariasis. Using this approach, we were able to predict stage-specific transcription factor binding sites involved in a stage of the molting process important for the establishment of the infection. We validated the role of these motifs using an in vitro molting system.

The tropical liver fluke, Fasciola gigantica is a food-borne parasite responsible for the hepatobiliary disease fascioliasis. The recent completion of F. gigantica genome sequencing by our group has provided a platform for the systematic analysis of the parasite genome. Eukaryotic protein kinases (ePKs) are regulators of cellular phosphorylation. In the present study, we used various computational and bioinformatics tools to extensively analyse the ePKs in F. gigantica (FgePKs) genome. A total of 455 ePKs were identified that represent ~2% of the parasite genome. Out of these, 214 ePKs are typical kinases (Ser/Thr- and Tyr-specific ePKs), and 241 were other kinases. Several FgePKs were found to possess unusual domain architectures, which suggests the diverse nature of the proteins that can be exploited for designing novel inhibitors. 115 kinases showed

Chimaeras, or ratfishes, are the only extant group of holocephalan fishes and are the sole host group of gyrocotylidean cestodes, which represent a sister group of the true tapeworms (Eucestoda). These unique, non-segmented cestodes have been known since the 1850s and multiple species and genera have been erected despite a general agreement that the delineation of species on the basis of morphology is effectively impossible. Thus, in the absence of molecular studies, the validity of gyrocotylid taxa and their specific host associations has remained highly speculative. Here we report the presence of Gyrocotyle spp. from rarely-caught deep-sea chimaeras collected in the North-East Atlantic, and describe two new species: G. haffii n. sp. from the bent-nose chimaera, Harriota raleighana Goode & Bean, and G. discoveryi n. sp. from the large-eyed rabbit fish, Hydrolagus mirabilis (Collett). Nuclear ribosomal sequence data were generated for individual parasites taken from different host species collected on different dates and from different localities and were combined with previously published sequences. Phylogenetic analyses supported the recognition of independent lineages and clusters, indicative of species, but were indecisive in recovering the root of the tree in analyses that included non-gyrocotylid outgroup taxa. The molecular data reveal variation not reflected in morphology and point to a complex picture of genetic divergence shaped by both isolation and migration in the deep-sea environment.

There is growing interest in local elimination of soil-transmitted helminth (STH) infection in endemic settings. In such settings, highly sensitive diagnostics are needed to detect STH infection. We compared double-slide Kato-Katz, the most commonly used copromicroscopic detection method, to multi-parallel quantitative polymerase chain reaction (qPCR) in 2,799 stool samples from children aged 2–12 years in a setting in rural Bangladesh with predominantly low STH infection intensity. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to double-slide Kato-Katz, STH prevalence using qPCR was almost 3-fold higher for hookworm species and nearly 2-fold higher for Trichuris trichiura. Ascaris lumbricoides prevalence was lower using qPCR, and 26% of samples classified as A. lumbricoides positive by Kato-Katz were negative by qPCR. Amplicon sequencing of the 18S rDNA from 10 samples confirmed that A. lumbricoides was absent in samples classified as positive by Kato-Katz and negative by qPCR. The sensitivity of Kato-Katz was 49% for A. lumbricoides, 32% for hookworm, and 52% for T. trichiura; the sensitivity of qPCR was 79% for A. lumbricoides, 93% for hookworm, and 90% for T. trichiura. Specificity was ≥ 97% for both tests for all STH except for Kato-Katz for A. lumbricoides (specificity = 68%). There were moderate negative, monotonic correlations between qPCR cycle quantification values and eggs per gram quantified by Kato-Katz. While it is widely assumed that double-slide Kato-Katz has few false positives, our results indicate otherwise and highlight inherent limitations of the Kato-Katz technique. qPCR had higher sensitivity than Kato-Katz in this low intensity infection setting., Author summary Soil-transmitted helminth infections (STH) (e.g., Ascaris, hookworm, Trichuris) contribute to a large burden of disease among children in low- and middle-income countries. There is increasing interest in implementing large-scale deworming programs to eliminate STH in certain settings. Efforts to monitor whether local elimination has occurred require sensitive diagnostic tests that will not miss positive cases. Kato-Katz, a microscopy-based diagnostic test, has commonly been used to identify STH eggs in stool, but in settings where infection intensity is low, this method frequently misses positive samples because it requires visual identification of small numbers of eggs, and hookworm eggs may degrade prior to visualization. Quantitative polymerase chain reaction (qPCR) is a molecular diagnostic method for detecting STH. It may detect more low intensity infections than Kato-Katz because it identifies STH DNA in stool; DNA can be detected in very small quantities and is less likely to degrade than STH ova. Thus, qPCR is likely to be more accurate than Kato-Katz. This study compared the performance of double-slide Kato-Katz and qPCR using 2,799 stool samples from children aged 2–12 years in a setting in rural Bangladesh with predominantly low STH infection intensity. qPCR was more sensitive than Kato-Katz for hookworm and Trichuris infections. 26% of samples were classified as Ascaris positive by Kato-Katz and negative by qPCR. DNA sequencing of 10 samples confirmed that Ascaris was absent in samples classified as positive by Kato-Katz and negative by qPCR. We conclude that Kato-Katz likely produced false positive results for Ascaris and that qPCR had a higher sensitivity than double-slide Kato-Katz in this low infection intensity setting.

Background Echinococcosis is a chronic zoonosis caused by tapeworms of the genus Echinococcus. Treatment of the disease is often expensive and complicated, sometimes requiring extensive surgery. Ultrasonographic imaging is currently the main technique for diagnosis, while immunological analysis provides additional information. Confirmation still needs pathological analysis. However, these diagnostic techniques generally detect infection in late stages of the disease. An accurate, early and non-invasive molecular diagnostic method is still unavailable. Methodology/Principal findings We sequenced the cell-free DNA (cfDNA) from plasma of echinococcosis patients and confirmed the presence of Echinococcus DNA. To improve detection sensitivity, we developed a method based on targeted next-generation sequencing of repeat regions. Simulation experiments demonstrate that the targeted sequencing is sensitive enough to detect as little as 0.1% of an Echinococcus genome in 1 mL of plasma. Results obtained using patient plasma shows that the Area Under the Curve (AUC) of the method is 0.862, with a detection sensitivity of 62.50% and specificity of 100%, corresponding to a Youden-index of 0.625. Conclusions/Significance This study provides evidence that hydatid cysts release cfDNA fragments into patient plasma. Using the repeat region targeted sequencing method, highly specific detection of Echinococcus infection was achieved. This study paves a new avenue for potential non-invasive screening and diagnosis of echinococcosis., Author summary Echinococcosis is a severe chronic parasitic disease caused by tapeworms of the genus Echinococcus. According to the World Health Organization, there are more than 1 million people living with echinococcosis worldwide. For decades, little progress has been made to develop a molecular diagnosis and specific treatment for the disease. Although imaging and immunological detection are used for diagnosis, these technologies are either only effective for late stages of the disease or hardly conclusive. The detection of cell-free DNA has been a powerful tool for precise diagnosis. In this study, we showed the presence of Echinococcus-derived cell-free DNA in plasma of echinococcosis patients. We further established an assay to detect parasite DNA in blood samples based on amplification of Echinococcus specific repeat regions followed by targeted next-generation sequencing. This technique provides a new method for potential extensive screening and precision diagnosis of echinococcosis with high specificity.

7 páginas, 2 tablas, 1 figura., The Stopping Transmission Of intestinal Parasites (STOP) project is part of the EDCTP2 programme supported by the European Union (grant number RIA2017NCT-1845 — STOP). JG is personally supported by Ramon Areces Foundation, Spain; MMV by the Spanish ‘Ramón y Cajal’ Programme, Ministry of Economy and Competitiveness (RYC-2015-18368); and SK is supported by DELTAS Africa Initiative grant # DEL15-011 to THRiVE-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In January and February 2019, a malacological survey was conducted in the area surrounding the residence of a 12-year-old child that had contracted cerebral angiostrongyliasis in the municipality of Macapá, capital of the Amapá State, northern Brazil. The serological examination was positive for Angiostrongylus cantonensis infection, the principal etiological agent of this parasitosis. A sample of 54 molluscs was artificially and individually digested for parasitological analysis, containing 38 specimens of Achatina fulica, nine specimens of Bulimulus tenuissimus and seven specimens of Sarasinula linguaeformis. A. fulica was the most abundant mollusc, and the only species infected with A. cantonensis, as well as presenting co-infections with other nematodes. This is the first report of cerebral angiostrongyliasis in the Amazon Region, and the first record of A. fulica infected with A. cantonensis in Amapá. These findings highlight the potential risks of human angiostrongyliasis, and the need to implement public health measures to control the spread of the disease.

Siganids are the most important marine fish distributed along the African coast. Therefore, the current study aimed to investigate parasite fauna infects one of the most important mariculture fish species in the Red Sea, the Rabbit fish Siganus rivulatus. One acanthocephalan species has been isolated from the posterior region of fish intestine, belonging to the Neoechinorhynchidae family, and named as Neoechinorhynchus macrospinosus Amin & Nahhas, 1994 based on its morphological and morphometric features. In order to determine the accurate taxonomic position of this acanthocephalan species, molecular phylogenetic analysis was carried out based on the partial sequences of 18S rDNA gene region. The obtained data revealed that this species was associated with a close identity ˃71% for other species belonging to the Neoechinorhynchidae family. In addition, the recovered species deeply embedded in the Neoechinorhynchus genus, closely related to the previously described Neoechinorhynchus sp., N. mexicoensis, and N. golvani with identity percent of 95.14, 93.59, 93.59%, respectively. Therefore, the present study provide a better understanding about the taxonomic status of N. macrospinosus based on 18S rDNA that can be useful for achieving a proper assessment of biodiversity. Resumo Os siganídeos são os peixes marinhos mais importantes distribuídos ao longo da costa africana. Portanto, o presente estudo teve como objetivo investigar a fauna de parasitas infectando uma das espécies mais importantes de peixes para maricultura no Mar Vermelho, o peixe-coelho Siganus rivulatus. Uma espécie de acantocéfalo foi isolada da região posterior do intestino de peixes pertencentes à família Neoechinorhynchidae, e denominadas Neoechinorhynchus macrospinosus Amin & Nahhas, 1994, com base em suas características morfológicas e morfométricas. A fim de determinar a posição taxonômica precisa dessa espécie de acantocéfalo, a análise filogenética molecular foi realizada com base nas sequências parciais da região do gene 18S rDNA e revelou que essa espécie estava associada a uma identidade próxima de até 71% para outras espécies pertencentes a família Neoechinorhynchidae e profundamente enraizada no gênero Neoechinorhynchus, intimamente relacionada a Neoechinorhynchus sp., N. mexicoensis, and N. golvani descrito anteriormente com percentual de identidade de 95,14, 93,59, 93,59%, respectivamente. Portanto, o presente estudo fornece uma melhor compreensão sobre o status taxonômico de N. macrospinosus com base no 18S rDNA que pode ser útil para obter uma avaliação adequada da biodiversidade.

Changes in parasite communities might result in new host-parasite dynamics and may threaten local fish populations. This phenomenon has been suggested for acanthocephalan parasites in the river Rhine and Danube where the species Pomphorhynchus tereticollis is becoming replaced by the Ponto-Caspian P. laevis. Developing knowledge on morphologic, genetic and behavioural differences between such species is important to follow such changes. However, disagreements on the current phylogeny of these two acanthocephalan species are producing conflicts that is affecting their correct identification. This study is offering a clearer morphological and genetic distinction between these two species. As P. tereticollis is found in rhithral tributaries of the Rhine, it was questioned whether the local salmonid populations were hosts for this species and whether P. laevis was expanding into the Rhine watershed as well. In order to test for this, brown trout, Salmo trutta, and grayling, Thymallus thymallus from South-Western Germany watersheds have been samples and screened for the occurrence of acanthocephalan parasites. For the first time, both species were confirmed to be hosts for P. tereticollis in continental Europe. P. tereticollis was found to be common, whereas P. leavis was found only at a single location in the Danube. This pattern suggest either that the expansion of P. laevis through salmonid hosts into rhithral rivers has not yet occurred, or that not yet ascertained biotic or abiotic features of rhithral rivers hinder P. laevis to spread into these areas.

Background The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. Methodology/principal findings We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. Conclusions/significance It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.

Ligophorus satunensis n. sp. was collected from the bluetail mullet, Crenimugil buchanani (Bleeker, 1853), caught off Satun, Thailand, representing the first report of the Ligophorus species in Thailand. The new species is most similar to Ligophorus fenestrum Soo & Lim, 2012 in its fenestrated dorsal hamuli, a ventral bar with a long, bifurcated anteromedian protuberance (AMP) without lateral pieces, and a non-sclerotized vagina. However, it differs from the latter in its dorsal hamuli (up to two layers of fenestrations of the dorsal hamuli in L. satunensis n. sp. vs six layers in L. fenestrum), ventral hamuli (nonfenestrated vs fenestrated), penis (turning a full circle before reaching the genital pore vs turning less than half a circle), and its accessory piece (non-grooved basally and grooved distally vs simply grooved along its entire length). A single specimen, very similar to L. satunensis n. sp., obtained from the same host fish, which can be differentiated from the latter by a considerably shorter AMP of the ventral bar and a smaller and slender dorsal hamuli, is tentatively designated here as Ligophorus sp. Moreover, the phylogenetic trees constructed from the concatenated sequences of partial large-subunit nuclear ribosomal DNA (28S) and nuclear ribosomal internal transcribed spacer 1 (ITS1) regions support that L. satunensis n. sp. is a new species closely related to L. fenestrum.

Schistosoma mansoni threatens hundreds of millions of people in >50 countries. Schistosomulae migrate through the lung and adult worms reside in blood vessels adjacent to the intestinal mucosa. Current candidate vaccines aren’t designed to elicit a mucosal response. We have repurposed an attenuated Salmonella enterica Typhimurium strain (YS1646) to produce such a vaccine targeting Cathepsin B (CatB), a digestive enzyme important for parasite survival. Promoter-Type 3 secretory signal pairs were screened for protein expression in vitro and transfected into YS1646 to generate candidate vaccine strains. Two strains were selected for in vivo evaluation (nirB_SspH1 and SspH1_SspH1). Female C57BL/6 mice were immunized twice, 3 weeks apart, using six strategies: i) saline gavage (control), ii) the ‘empty’ YS1646 vector orally (PO) followed by intramuscular (IM) recombinant CatB (20μg IM rCatB), iii) two doses of IM rCatB, iv) two PO doses of YS1646-CatB, v) IM rCatB then PO YS1646-CatB and vi) PO YS1646-CatB then IM rCatB. Serum IgG responses to CatB were monitored by ELISA. Three weeks after the second dose, mice were challenged with 150 cercariae and sacrificed 7 weeks later to assess adult worm and egg burden (liver and intestine), granuloma size and egg morphology. CatB-specific IgG antibodies were low/absent in the control and PO only groups but rose substantially in other groups (5898-6766ng/mL). The highest response was in animals that received nirB_SspH1 YS1646 PO then IM rCatB. In this group, reductions in worm and intestine/liver egg burden (vs. control) were 93.1% and 79.5%/90.3% respectively (all P < .0001). Granuloma size was reduced in all vaccinated groups (range 32.9–52.8 x103μm2) and most significantly in the nirB_SspH1 + CatB IM group (34.7±3.4 x103μm2vs. 62.2±6.1 x103μm2: vs. control P < .01). Many eggs in the vaccinated animals had abnormal morphology. Targeting CatB using a multi-modality approach can provide almost complete protection against S. mansoni challenge., Author summary Schistosomiasis is a parasitic disease that affects over 250 million people worldwide and over 800 million are at risk of infection. Of the three main species, Schistosoma mansoni is the most widely distributed and is endemic in the Caribbean, South America, and Africa. It causes a chronic disease with severe negative effects on quality of life. Mass drug administration of praziquantel is the only available course of action due to a current lack of vaccines. However, praziquantel does not protect from reinfection. Therefore, a vaccine would be beneficial as a long-term solution to reduce morbidity and transmission of the disease. Our group has repurposed the attenuated YS1646 strain of Salmonella Typhimurium as an oral vaccine vector for the digestive enzyme Cathepsin B of S. mansoni. Oral vaccination followed by an intramuscular dose of recombinant Cathepsin B lead to significant reductions in parasite burden in mice. These animals had the highest titers in serum IgG and intestinal IgA antibodies. This multimodal vaccination approach also elicited both Th1 and Th2 cytokines as seen by the increases in IFNγ and IL-5. Finally, vaccinated mice had reductions in granuloma size along with a higher proportion of morphologically-abnormal eggs. This work demonstrates that a YS1646-based, multimodality, prime-boost immunization schedule can provide nearly complete protection against S. mansoni in a well-established murine model.

The work is part of the EU ERA-Net program BiodivERsA, under the project BioMarKs (Biodiversity of Marine euKaryotes).-- 14 pages, 8 figures, supplementary material https://doi.org/10.1038/s41598-018-27509-8, Although animals are among the best studied organisms, we still lack a full description of their diversity, especially for microscopic taxa. This is partly due to the time-consuming and costly nature of surveying animal diversity through morphological and molecular studies of individual taxa. A powerful alternative is the use of high-throughput environmental sequencing, providing molecular data from all organisms sampled. We here address the unknown diversity of animal phyla in marine environments using an extensive dataset designed to assess eukaryotic ribosomal diversity among European coastal locations. A multi-phylum assessment of marine animal diversity that includes water column and sediments, oxic and anoxic environments, and both DNA and RNA templates, revealed a high percentage of novel 18S rRNA sequences in most phyla, suggesting that marine environments have not yet been fully sampled at a molecular level. This novelty is especially high among Platyhelminthes, Acoelomorpha, and Nematoda, which are well studied from a morphological perspective and abundant in benthic environments. We also identified, based on molecular data, a potentially novel group of widespread tunicates. Moreover, we recovered a high number of reads for Ctenophora and Cnidaria in the smaller fractions suggesting their gametes might play a greater ecological role than previously suspected, This work was supported by an Institució Catalana de Recerca i Estudis Avançats contract, two grants (BFU-2011-23434 and BFU2014-57779-P) from the Ministerio de Economia y Competitividad (MINECO), one of which (BFU2014-57779-P) was co-funded by the European Regional Development Fund (FEDER), and a European Research Council Consolidator Grant (ERC-2012-Co -616960) to I.R.-T. We also acknowledge financial support from the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (Project 2014 SGR 619). J.d.C. was supported by a Marie Curie International Outgoing Fellowship grant (FP7-PEOPLE-2012-IOF - 331450 CAARL). J.P. acknowledges support from the European Research Council under the European Union’s Seventh Framework Program (FP7/2007–2013)/ERC grant [268513]

Defining the optimal diagnostic tools for evaluating onchocerciasis elimination efforts in areas co-endemic for other filarial nematodes is imperative. This study compared three published polymerase chain reaction (PCR) methods: the Onchocerca volvulus-specific qPCR-O150, the pan-filarial qPCR melt curve analysis (MCA), and the O150-PCR enzyme-linked immunosorbent assay (ELISA) currently used for vector surveillance in skin snip biopsies (skin snips) collected from the Democratic Republic of the Congo. The pan-filarial qPCR-MCA was compared with species-specific qPCRs for Loa loa and Mansonella perstans. Among the 471 skin snips, 47.5%, 43.5%, and 27.0% were O. volvulus positive by qPCR-O150, qPCR-MCA, and O150-PCR ELISA, respectively. Using qPCR-O150 as the comparator, the sensitivity and specificity of qPCR-MCA were 89.3% and 98.0%, respectively, whereas for O150-PCR ELISA, they were 56.7% and 100%, respectively. Although qPCR-MCA identified the presence of L. loa and Mansonella spp. in skin snips, species-specific qPCRs had greater sensitivity and were needed to identify M. perstans. Most of the qPCR-MCA misclassifications occurred in mixed infections. The reduced sensitivity of O150-PCR ELISA was associated with lower microfilaria burden and with lower amounts of O. volvulus DNA. Although qPCR-MCA identified most of the O. volvulus-positive skin snips, it is not sufficiently robust to be used for stop-mass drug administration (MDA) evaluations in areas co-endemic for other filariae. Because O150-PCR ELISA missed 43.3% of qPCR-O150-positive skin snips, the qPCR-O150 assay is more appropriate for evaluating skin snips of OV-16 + children in stop-MDA assessments. Although improving the sensitivity of the O150-PCR ELISA as an alternative to qPCR might be possible, qPCR-O150 offers distinct advantages aside from increased sensitivity.

Four strains of entomopathogenic nematodes were isolated with a live trap method in southern Poland. The isolates were identified asSteinernema silvaticumbased on morphological, morphometric and molecular data. Infective juveniles of PolishS. silvaticumisolates differ in body length fromS. weiseri(951 vs 740 μm, respectively), and in the hyaline tail portion fromS. kraussei(48 vs 38%, respectively). First-generation males ofS. silvaticumare longer than those ofS. kraussei,S. weiseriandS. ichnusae(1829 vs 1400, 1180 and 1341 μm, respectively). Males ofS. silvaticumand a sister speciesS. krausseican be distinguished by the distance from the anterior end to the nerve ring (142 vs 105 μm), spicule (66 vs 49 μm) and gubernaculum length (45 vs 33 μm), and the presence of a mucron. The analysis of internal transcribed spacer (ITS), D2-D3 andcox1sequences of the tested nematodes revealed differences of 3–5%, 3% and 12–13%, respectively, fromS. krausseistrains. The phylogeny of both nuclear and mitochondrial genes indicated close relationships of the PolishS. silvaticumisolates withS. kraussei,S. oregonenseandS. cholashanense. The reproductive isolation of the studied isolates was confirmed by hybridization tests with other Europeanfeltiae–krausseigroup representatives. This study has supplemented the original description ofS. silvaticumwith morphological and morphometric characterization of the first-generation males and females. This is also the first molecular study of this species based on a multi-gene approach.

RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition., The C. elegans LIN-41 and its homologs, including human TRIM71/LIN41, contain the RNA binding NHL domain. Here the authors combine computational analysis, structural biology and in vivo studies, to explain how these proteins bind RNA and how rapid evolution of NHL domains resulted in different solutions to RNA recognition.

Aphelenchoides besseyi and A. fujianensis have been frequently found in mixed populations associated with forage grass seed in Brazil. The morphological similarity between both species has previously led A. fujianensis to be erroneously identified as A. besseyi. A. besseyi is a quarantine pest in many countries that import Brazilian forage seed; however, there is no current evidence suggesting that A. fujianensis is a plant-parasitic species. Two real-time polymerase chain reaction (qPCR) diagnostics were developed to detect each species and an operational envelope was established. A set of primers and hydrolysis probes for each species was designed targeting the large subunit (LSU) region. To assess their specificity, primers and probes sets were tested with samples of nontarget Aphelenchoides and Paraphelenchus sp. also frequently associated with forage seed. Experiments using dilutions of purified plasmid standards underpinned the sensitivity of the qPCR assays, which detected as few as 10 copies of target nematode ribosomal DNA. Thus, the developed diagnostics were sufficiently sensitive to detect DNA extracted from a fragment of a single target nematode. There was a positive correlation between copy number of the target species and nematode abundance, suggesting the potential of this method for quantification. Evidence of intra-individual variability among cloned sequences of the LSU region in a single A. besseyi population is also reported.

Echinococcus granulosus is a helminth from the family Taeniidae, which causes cystic echinococcosis (CE) in humans and diverse livestock around the world. The identification of existing genotypes in different regions is a major step towards the prevention and establishment of control programmes for the disease. This study aimed to detect CE genotypes using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the internal transcribed spacer-1 (ITS1) gene and sequencing of the cytochrome c oxidase subunit 1 (Cox1) gene in isolates from the central part of Mazandaran province, northern Iran. Forty isolates were collected from sheep, 17 from cattle and 6 from human formalin-fixed paraffin-embedded tissues (FFPE). The ITS1 and Cox1 genes were successfully amplified by PCR in 41 and 42 samples, respectively. PCR-RFLP and sequencing showed that all isolates had the G1–G3 genotypes in this study. Out of 31 isolates subjected to sequencing for the Cox1 gene, 80.7% had the G1 genotype. G2 (16.1%) and G3 (3.2%) genotypes were observed in five sheep and one cattle samples, respectively. Five human isolates were also sequenced for the ITS1 gene, which showed that all samples belonged to the G1 genotype. Ten haplotypes were determined among the isolates by alignment analysis of the Cox1 gene. In summary, this study demonstrated that G1 was the dominant genotype circulating between humans and livestock in the studied region. Furthermore, high genotypic diversity among the CE isolates was observed.

Self-defense by avoiding self-targeting By silencing transposons, Piwi-interacting RNAs (piRNAs) protect the stability of animal genomes in germ lines. However, many piRNAs do not map to transposons, and their functions have remained undefined. Zhang et al. described the piRNA targeting logic in Caenorhabditis elegans and identified an intrinsic sequence signal in endogenous germline genes that confer resistance to piRNA silencing. Thus, diverse piRNAs silence foreign nucleic acids but spare self genes to defend the C. elegans genome. In addition, multiple foreign transgenes can be engineered to escape piRNA targeting, allowing successful expression in the germline. Science , this issue p. 587

Adults of Hysteromorpha triloba (Rudolpi, 1819), Lutz, 1931 inhabit primarily the intestine of cormorants across the globe, whereas metacercariae have been found in the body cavity of freshwater fishes of the families Cyprinidae, Ictaluridae, Ariidae, Pimelodidae and Catostomidae. In this study, adults and metacercariae identified as H. triloba were collected from the Neotropical cormorant (Nannopterum brasilianus) and from the Mexican tetra fish (Astyanax mexicanus) from the Gulf of Mexico and Pacific Ocean slopes in the Neotropical region. Partial DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I (cox 1) and the internal transcribed spacers (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA were generated for both developmental stages, and were compared with available sequences of H. triloba from the Nearctic region. The genetic divergence between metacercariae and adults of H. triloba from the Neotropical and Nearctic region (Canada) associated with the double-crested cormorant (Nannopterum auritus), ranged from 0 to 5.5% for cox 1 and from 0 to 0.2% for ITS. Phylogenetic analyses inferred with both molecular markers using maximum likelihood and Bayesian inference placed the adults and metacercariae in a single clade, confirming that both stages are conspecific. Our data confirmed that H. triloba is a widely distributed species across the Americas, parasitizing both the Neotropical and Nearctic cormorants in Argentina, Brazil, Venezuela, Mexico, USA and Canada.

To develop a Tm-shift method for detection of dog-derived Ancylostoma ceylanicum and A. caninum, three sets of primers were designed based on three SNPs (ITS71, ITS197, and ITS296) of their internal transcribed spacer 1 (ITS1) sequences. The detection effect of the Tm-shift was assessed through the stability, sensitivity, accuracy test, and clinical detection. The results showed that these three sets of primers could distinguish accurately between A. ceylanicum and A. caninum. The coefficient of variation in their Tm values on the three SNPs was 0.09% and 0.15% (ITS71), 0.18% and 0.14% (ITS197), and 0.13% and 0.07% (ITS296), respectively. The lowest detectable concentration of standard plasmids for A. ceylanicum and A. caninum was 5.33 × 10−6 ng/μL and 5.03 × 10−6 ng/μL. The Tm-shift results of ten DNA samples from the dog-derived hookworms were consistent with their known species. In the clinical detection of 50 fecal samples from stray dogs, the positive rate of hookworm detected by Tm-shift (42%) was significantly higher than that by microscopic examination (34%), and the former can identify the Ancylostoma species. It is concluded that the Tm-shift method is rapid, specific, sensitive, and suitable for the clinical detection and zoonotic risk assessment of the dog-derived hookworm.

Filarial infections of humans cause some of the most important neglected tropical diseases. The global efforts for eliminating filarial infections by mass drug administration programs may require additional tools (safe macrofilaricidal drugs, vaccines, and diagnostic biomarkers). The accurate and sensitive detection of viable parasites is essential for diagnosis and for surveillance programs. Current community-wide treatment modalities do not kill the adult filarial worms effectively; hence, there is a need to identify and develop safe macrofilaricidal drugs. High-throughput sequencing, mass spectroscopy methods and advances in computational biology have greatly accelerated the discovery process. Here, we describe post-genomic developments toward the identification of diagnostic biomarkers and drug targets for the filarial infection of humans.

Background Human clonorchiasis, caused by the infection of Clonorchis sinensis, is one of the major health problems in Southeast Asia. However, vaccine efficacy against C. sinensis infection remains largely unknown. Methods In this study, for the first time, we generated virus-like particles (VLPs) vaccine containing the C. sinensis tegumental protein 22.3 kDa (CsTP 22.3) and the influenza matrix protein (M1) as a core protein, and investigated the vaccine efficacy in Sprague-Dawley rats. Results Intranasal immunization of VLPs vaccine induced C. sinensis-specific IgG, IgG2a and IgG2c in the sera and IgA responses in the feces and intestines. Notably, upon challenge infection with C. sinensis metacercariae, significantly lower adult worm loads (70.2%) were measured in the liver of rats immunized with VLPs, compared to those of naïve rats. Furthermore, VLPs immunization induced antibody secreting cells (ASC) responses and CD4+/CD8+ T cell responses in the spleen. Conclusions Our results indicated that VLPs vaccine containing C. sinensis CsTP 22.3 kDa provided partial protection against C. sisnensis infection. Thus, VLPs could be a potential vaccine candidate against C. sinensis. Electronic supplementary material The online version of this article (10.1186/s13071-017-2526-5) contains supplementary material, which is available to authorized users.

Ribosomal protein S9 (RPS9) is an essential functional gene that participates in DNA repair and developmental regulations. A sequence homolog of RPS9 has been found to be upregulated in the protoscoleces (PSCs) of Echinococcus granulosus treated with artemisinin. However, E. granulosus RPS9 (EgRPS9) has not been identified before. In the present study, the 657-base pair (bp) cDNA encoding EgRPS9 was cloned. Amino acid sequence analysis showed that EgRPS9 was similar to the RSP9 proteins from Schistosoma japonicum (SjRPS9, 86%) and Schistosoma mansoni (SmRPS9, 79%). Phylogenetic tree analysis showed that EgRPS9, SmRPS9, and SjRPS9 were clustered together. We detected the EgRPS9 gene and protein expression in PSCs exposed to artesunate (AS) which displayed a dose-dependent reduction in PSC viability for 24 hr. The results showed that the EgRPS9 ratio of the 10-μM AS-treated ( P0.01) and 40-μM AS-treated ( P0.05) groups were increased from that of the control group. In addition, the level of reactive oxygen species (ROS) in the AS-treated groups increased in a dose-dependent manner compared to the level in the control group. In conclusion, the expression of EgRPS9 could be induced by ROS and might participate in the oxidative damage-based anti-parasite mechanism of AS treatment.

Adult Aphanurus mugilis Tang, 1981 worms were detected in the intestine of Moolgarda engeli in the shallow waters off Cat Ba Island, Vietnam. Tang (1981) first described this species in Mugil cephalus off China. The worms in Vietnamese mullet were identical to Chinese specimens in a number of morphometric characteristics, with the exception of body and ovary size. In the present study, morphological characteristics, and the first molecular data for A. mugilis are provided. Additionally, molecular phylogenetic analysis of the family Hemiuridae was performed. The results of our molecular phylogenetic study indicate that the presence or absence of an ecsoma was not associated with molecular data for hemiurid subfamilies differentiation. The basal position of Bunocotylinae on the molecular-based phylogenetic tree indicated a primordial nature of ecsoma of hemiurid trematodes. Considerable molecular differentiation of Bunocotylinae from other hemiurids indicated the possibility of the recognition of the family Bunocotylidae Dollfus, 1950. Assuming that Machidatrema chilostoma is considered within the Bunocotylinae, the paraphyly of the Lecithasterinae was supported.

A new species belonging to the genus Belondira is described from natural areas in Iran. Belondira coomansi n. sp. is characterized by its general size, the dimensions and appearance of its lip region, presence of distinct labial and post-labial sclerotization, the length of the odontostyle and its inconspicuous lumen and aperture, the length of the neck and its pharyngeal expansion, the reduction of the female anterior genital branch to a simple uterine sac, a very short posterior uterus, the anterior position of the vulva, the length and shape of the caudal region with distinctly thick cuticle at its tip, the length of the spicules, and the presence of only one pair of ventromedian supplements. The new species is close to Belondira brevibulba, B. sacchari, B. tenuidens and B. thornei, and it is compared to them. Molecular characterization (D2–D3 expansion segments of the rRNA large subunit) of the new species is also provided, representing only the second species of this genus for which any DNA sequence data are available.

Timoniellaspp. are cryptogonimid flukes (Digenea: Cryptogonimidae) that parasitize the guts of fish in brackish waters.Timoniella imbutiforme, a species from the Mediterranean Sea, is recorded in the Black Sea, whileT. balthicahas been described from the Baltic Sea. In this paper, we clarify the taxonomic status ofTimoniellapopulations in the Baltic and Black seas. Adults and metacercariae ofTimoniellaspp. were sampled from localities in the Mediterranean Sea (France), Black Sea (Ukraine) and Baltic Sea (Germany) and subjected to molecular and morphological analysis, including Bayesian phylogenetic reconstruction based on concatenated sequences of ITS1–ITS2–28S. This allowed us to construct a new key to species of the genusTimoniella. Our results suggest thatT. balthicaforms part of the Boreal–Atlantic relict fauna of the Black Sea and should now be considered a junior synonym ofT. imbutiforme.

The diversity and taxonomy of diplostomids infecting freshwater fishes of the Indian region are predominantly poorly known. Yet compared to other trematodes, identification and discrimination of diplostomid metacercaria are difficult using morphology and site of infection. The present study provides the first molecular study of a Tylodelphys sp. from India. Specimens of Tylodelphys were collected from the vitreous humor of the eyes and from the brains of siluriform fish, Mystus tengara (Siluriformes: Bagridae) from Hastinapur, Meerut (U.P.), India. Taxonomic studies were performed on the basis of morphological, morphometrical, and molecular analysis. The dissimilarities in morphological and sites of infection between 2 forms of Tylodelphys, designated as Tylodelphys sp. 1 and Tylodelphys sp. 2, suggested that the forms were different species but, after performing molecular analysis, we conclude that despite morphological differences both morphotypes are conspecific.

Caenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development. In contrast to ALG-1 and ALG-2, which associate with most miRNAs and have general roles throughout development, ALG-5 interacts with only a small subset of miRNAs and is specifically expressed in the germline where it localizes alongside the piRNA and siRNA machinery at P granules. alg-5 is required for optimal fertility and mutations in alg-5 lead to a precocious transition from spermatogenesis to oogenesis. Our results provide a near-comprehensive analysis of miRNA-Argonaute interactions in C. elegans and reveal a new role for miRNAs in the germline.

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing.

Ancylostoma tubaeformemay infect canids, felids and humans, and pose a potential risk to public health. Polymerase chain reaction (PCR) techniques were used to amplify the complete mitochondrial (mt) genome sequence ofA. tubaeformefrom cats and to analyse its sequence characteristics after molecular identification based on the internal transcribed spacer ITS1+ sequence. The results show that the complete mt genome sequence (GenBank accession number KY070315) ofA. tubaeformefrom cats was 13,730 bp in length, including 12 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, two non-coding regions and an AT-rich region. The nucleotide content of A and T was 77.93%, biased toward A and T. Twelve protein-coding genes used ATT, TTG and GTG as initiation codons, and TAA, TAG, TA and T as termination codons. The length of the 22 tRNA genes ranged from 52 to 62 bp, their predicted secondary structures were D loops and V loops. The lengths of the two rRNAs were 958 and 697 bp. Phylogenetic analyses showed thatA. tubaeformefrom cats was in the lineage ofAncylostoma, having a close phylogenetic relationship withA. caninum. This study reports for the first time the mt genome ofA. tubaeformefrom cats in China, which could enhance the mt genome database of Ancylostomatidae nematodes, and it offers the scientific basis for further studies in the genetic diversity of hookworms among different hosts.

The RNA interference (RNAi) has the ability to turn off individual gene expression. So, it affords a remarkably specific tool for studying the effects of genes. It is regarded as a direct approach for determining such gene/genes functions and offers a valuable tool for modern drug discovery. The study aimed to develop in vitro RNAil in Brugia malayi with particular interest to study the function of Brugia malayi avr-14 (Bm-avr-14) and Brugia malayi f-tubulin (Bm-fi-tubulin) genes. Bm-avr-14 is a gene encoding glutamate gated chloride channel (GluCl) which binds ivermectin and Bm-β-tubulin is a gene encoding β-tubulin which binds albendazole. Adult female worms were soaked in heterogeneous short interfering RNA (hsiRNA) with interest to study the role of two genes Bm-avr-14 and Bm-β-tubulin. Then, we assessed the knock down effects of target genes using worminator system and real time PCR. We found that worms treated with hsiRNA of Bm-avr-14 had a significant reduction in microfilariae (mf) production in comparison with untreated worms or worms treated with hsiRNA of green fluorescent protein (GFP). No significant reduction in mf production with Bm-β-tubulin gene was obtained. There were no changes in the movement of adults treated with either Bm-avr-14 or Bm- β-tubulin hsiRNAs. Inconsistent RNAi mediated suppression was achieved with Bm-avr-14 and Bm-β- tubulin using real time PCR. The data may be helpful in assessment of drug target potential of genes.

Paramphistomosis, caused by paramphistomid flukes, is a gastrointestinal parasitic disease of domestic and wild ruminants. Originally thought to be limited to the tropics and subtropics, the disease has recently been reported in temperate regions. Here we describe the concurrent infection of a red deer doe (Cervus elaphus) with Paramphistomum leydeni and Paramphistomum cervi. This is the first report of P. leydeni in Croatia. Flukes were identified on the basis of morphological keys (tegumental papillae) and sequencing of the internal transcribed spacer region 2 in ribosomal DNA. Our results confirm that the absence of tegumental papillae allows P. cervi to be differentiated morphologically from other paramphistomid species in Europe based on incident light stereomicroscopy. Nevertheless the limitations of morphological identification and taxonomic issues suggest that previous findings on paramphistomid infection should be interpreted carefully. The possible worldwide distribution of these pathogens means that paramphistomosis may be more common and its economic impact greater than previously thought.