Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.

This study investigated Echinococcus genotypes in patients with hydatidosis that reside in Inner Mongolia, Tibet or Qinghai Province by partially sequencing the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) genes. Cyst fluids were collected from 23 patients with hydatidosis and DNA was extracted. Portions of the cox1 and nad1 genes were amplified and subsequently sequenced. Sequencing analysis determined that one of the isolates contained Echinococcus multilocularis, and the other 22 isolates contained E. granulosus sensu lato. The isolates were then further classified based on genotype, and E. granulosus sensu stricto (s.s.) G1 (n = 20), E. granulosus s.s. G3 (n = 1) and E. canadensis G6/7 (n = 1) were identified. Additionally, the sequences were concatenated (pcox1 + pnad1) and 11 haplotypes were identified among the E. granulosus s.s. isolates (G1 and G3), with a shared common haplotype (H1) identified. Overall, these findings provide further understanding of the genetic patterns of Echinococcus in western and northern China.

Abdominal angiostrongyliasis is an endemic zoonosis in southern Brazil caused by the nematode Angiostrongylus costaricensis, which uses terrestrial molluscs as intermediate hosts and wild rodents as final hosts. Humans can be infected by ingesting infectious A. costaricensis larvae. To date, correlations between shedding of first-stage larvae (L1) and different infective doses of third-stage larvae (L3) have not been elucidated. The aim of this study was to assess L1 faecal shedding levels in Swiss mice experimentally infected with different doses of A. costaricensis L3 and to determine whether infective doses are related to mortality. Thirty-two male Swiss mice were divided evenly into a non-infected control (NI-Con); low-dose infection (LD-Inf); medium-dose infection (MD-Inf) and high-dose infection (HD-Inf) groups infected with 0, 5, 15 and 30 A. costaricensis L3, respectively. Faecal samples were collected from each animal, starting at day 20 post infection. HD-Inf mice had greater faecal L1 shedding levels than LD-Inf mice, but not a significantly shortened survival. In conclusion, infective doses of A. costaricensis L3 affect L1 shedding levels without altering mortality in Swiss mice.

Dawestrema cycloancistrium is the main ectoparasite causing mortality in fingerlings of pirarucu (Arapaima gigas) in Amazonian aquaculture. Very little is known about the D. cycloancistrium reproductive index and appropriate methods of collecting eggs for investigation. This study aimed to determine the oviposition rate of D. cycloancistrium. To achieve this aim, two egg quantification methods were tested: the estimative method (ME) and the total counting method (MT). Compared with the MT, the ME overestimated the number of eggs counted, which were 2943.5 ± 2840.6 and 1041.5 ± 533 eggs, and the oviposition rate, which was 80.1 ± 58.7 and 31.4 ± 16.4 eggs/parasite/day, for ME and MT, respectively. These results show that for studies quantifying D. cycloancistrium eggs, the total eggs in the sample must be counted, as the estimates made using subsamples are not representative. Using the MT, the oviposition rate for D. cycloancistrium was determined to be 31.4 ± 16.4 eggs per adult parasite per day. The present study demonstrates the egg production capacity of a monogenean species parasite of A. gigas, providing basic biological data for D. cycloancistrium.

A total of 1840 brine shrimps (Artemia franciscana) were examined for cestode larvae at monthly intervals between November 2015 and June 2016. Of these, 663 (36.03%) specimens were infected with cysticercoids of seven cestode species in numbers between one and sixteen. During the first four months of examination, the percentage of infected shrimps was low but rose significantly with increasing temperatures in March, reaching maximum values in May. Flamingolepis liguloides and Flamingolepis flamingo showed the highest prevalence overall, at 25.3 and 10.7%, respectively. The intensity of infection was 1–10 and 1–4 cysticercoids, respectively. Eurycestus avoceti, Wardium stellorae, Gynandrotaenia stammeri, Anomotaenia tringae and Confluaria podicipina occurred at lower prevalence of 4.5, 3.2, 1.7, 0.3 and 0.05%, respectively. Up to four species were detected in one host.

This study focused on the spirurid nematode Mastophorus muris in water voles (Arvicola amphibius) trapped in three regions in southern Sweden during spring and fall 2013. The collection of water voles formed part of a larger project (EMIRO) on the cestode Echinococcus multilocularis in rodents. The voles’ stomach contents were examined for the presence of M. muris. Prevalence, mean abundance and mean intensity of infection were calculated. A generalized linear model model was used to examine the effects of sex, functional group, season and region on the number of M. muris individuals in each vole. Forty-seven of 181 (26%) voles were infected with M. muris, with up to 74 worms each. The overall mean intensity (worms per infected vole) was 15 (95% CI 10–21), and abundance (mean number of worms in all voles) was 4 (95% CI 2–6). Model output indicated a significant effect of season and region with respect to abundance of nematode infection, which was independent of sex and functional group of the investigated host.

Enterobiasis, caused by the nematode Enterobius vermicularis, is a common health problem among schoolchildren in Thailand. We provide the first molecular identification of this nematode from Thai schoolchildren and document genetic variation among E. vermicularis eggs using sequence analyses of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). A cross-sectional parasitological survey was conducted in schoolchildren (n = 491) in five regions of Thailand between May 2015 and December 2016. The diagnosis of Enterobius infection was made using the adhesive tape perianal swab technique. Enterobius eggs were recovered from 43 participants (8.75%). DNA was extracted from these eggs and the cox1 gene and partial ITS2 region amplified using the polymerase chain reaction (PCR). Nineteen amplified PCR products of the cox1 gene (441 bp) and 18 of the ITS2 region (623 bp) were subsequently sequenced. All sequences were identified as belonging to E. vermicularis based on database searches. Phylogenetic analysis and a median-joining network of available E. vermicularis cox1 sequences showed 66 haplotypes. We found haploclusters (types A and B) represented among the Thai sequences. Six haplotypes from Thailand fell into type A (of Nakano et al., 2006) (along with sequences from Japan and Korea) and five haplotypes into type B (with sequences from Japan, Iran, Czech Republic, Greece, Denmark and Sudan). The overall haplotype diversity (Hd) was 0.9888. Transmission of worms with type B haplotypes from primates to humans in Asia or from humans in Europe possibly occurs in Thailand.

Hookworms of the genus Uncinaria parasitize pinniped pups in various locations worldwide. Four species have been described, two of which parasitize pinniped pups in the southern hemisphere: Uncinaria hamiltoni parasitizes Otaria flavescens and Arctocephalus australis from the South American coast, and Uncinaria sanguinis parasitizes Neophoca cinerea from the Australian coast. However, their geographical ranges and host specificity are unknown. Uncinaria spp. are morphologically similar, but molecular analyses have allowed the recognition of new species in the genus Uncinaria. We used nuclear genetic markers (internal transcribed spacer (ITS) and large subunit (LSU) rDNA) and a mitochondrial genetic marker (cytochrome c oxidase subunit I (COI)) to evaluate the phylogenetic relationships of Uncinaria spp. parasitizing A. australis and O. flavescens from South American coasts (Atlantic and Pacific coasts). We compared our sequences with published Uncinaria sequences. A Generalized Mixed Yule Coalescent (GMYC) analysis was also used to delimit species, and principal component analysis was used to compare morphometry among Uncinaria specimens. Parasites were sampled from A. australis from Peru (12°S), southern Chile (42°S), and the Uruguayan coast, and from O. flavescens from northern Chile (24°S) and the Uruguayan coast. Morphometric differences were observed between Uncinaria specimens from both South American coasts and between Uncinaria specimens from A. australis in Peru and southern Chile. Phylogenetic and GMYC analyses suggest that south-eastern Pacific otariid species harbour U. hamiltoni and an undescribed putative species of Uncinaria. However, more samples from A. australis and O. flavescens are necessary to understand the phylogenetic patterns of Uncinaria spp. across the South Pacific.

Phasmarhabditis hermaphrodita is a parasitic nematode of terrestrial gastropods that has been formulated into a biological control agent for farmers and gardeners to kill slugs and snails. In order to locate slugs it is attracted to mucus, faeces and volatile cues; however, there is no information about whether these nematodes are attracted to snail cues. It is also unknown how wild isolates of P. hermaphrodita or different Phasmarhabditis species behave when exposed to gastropod cues. Therefore, we investigated whether P. hermaphrodita (commercial and wild isolated strains), P. neopapillosa and P. californica were attracted to mucus from several common snail species (Cepaea nemoralis, Cepaea hortensis, Arianta arbustorum and Cornu aspersum). We also examined whether snails (C. aspersum) collected from different locations around the UK differed in their attractiveness to wild isolates of P. hermaphrodita. Furthermore, we also investigated what properties of snail mucus the nematodes were attracted to, including hyaluronic acid and metal salts (FeSO4, ZnSO4, CuSO4 and MgSO4). We found that the commercial strain of P. hermaphrodita responded poorly to snail mucus compared to wild isolated strains, and C. aspersum collected from different parts of the UK differed in their attractiveness to the nematodes. We found that Phasmarhabditis nematodes were weakly attracted to all metals tested but were strongly attracted to hyaluronic acid. In a final experiment we also showed that pharmacological manipulation of cyclic guanosine monophosphate (cGMP) increased chemoattraction to snail mucus, suggesting that the protein kinase EGL-4 may be responsible for Phasmarhabditis sp. chemoattraction.

Despite the reduction in the prevalence of soil-transmitted helminthiases in many regions of the world, morbidity rates remain high in some rural regions. The Kato–Katz technique is a simple, inexpensive and field-applicable tool commonly used for the diagnosis and worm-burden characterization of these infections. Molecular studies have revolutionized our understanding of the epidemiology and evolutionary genetics of parasites. In this study we recovered helminthic DNA from Kato–Katz slides (n = 93) prepared in 2011 in the Brazilian Amazon. We achieved DNA recovery by polymerase chain reaction (PCR) in 84% of cases for Ascaris sp. and 75% of cases for hookworms. The sequencing confirmed the specific species of the amplicons. The slides stored for a few years could be analysed using this methodology, allowing access to DNA from a large collection of samples. We must consider the Kato–Katz thick smears as a source of helminth DNA. This can significantly reduce logistical difficulties in the field in terms of obtaining, preserving, transporting and initial processing of samples.

The spiral intestines of a total of 16 round fantail stingrays Taeniurops grabata from the Mediterranean Sea off Tunisia were examined for cestodes. A new genus is erected in the Anthocephaliidae (Rhinebothridea) as Alveobothrium gen. n., with Alveobothrium grabatum sp. n. as its type species; the new genus differs from the other genera in the order in that its members possess bothridia with an apical sucker, marginal loculi and multiple staggered rows of facial loculi. Alveobothrium zarzisense sp. n. is also described. The species differ in the number of marginal loculi and in proglotid anatomy. Another anthocephaliid belonging to the genus Anthocephalum is also described from T. grabata. Anthocephalum jeancadenati sp. n. is most similar to A. alicae and A. michaeli, but differs in size of terminal proglottid and number of proglottids. All these new species are also found in formalin-preserved cestodes from T. grabata collected at Gorée Island (Senegal) between 1946 and 1954 by the French ichthyologist J. Cadenat and conserved in the personal collection of the late L. Euzet. The presence of the same rhinebothriideans species parasitizing T. grabata in both the Mediterranean (Tunisia) and the eastern Atlantic (Senegal) is discussed.

Uveitis is one of the commonest causes of vision loss worldwide and its exact etiology is still not clarified in most patients. The current study is a trial to assess the efficacy of serum anti-Toxocara immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for ocular toxocariasis (OT) and to detect OT prevalence and the associated ocular manifestations in sera of patients with uveitis. One hundred and twelve patients (62 females and 50 males) with uveitis were diagnosed by ophthalmologists, radiologists and rheumatologists according to ocular manifestations, laboratory and radiological investigations. Serum anti-Toxocara IgG titers were determined by ELISA in sera of all patients. Our results revealed that OT is highly associated with intermediate and posterior uveitis. Children and young adult females, especially those residing in rural areas, complained mainly of diminution of vision in the left eye, with strabismus and leukocoria. At a cut-off value of 0.258, the sensitivity and specificity of IgG ELISA were 93.3% and 100%, respectively. In conclusion, at a novel cut-off value of 0.258 the serum anti-Toxocara IgG ELISA is predicted to be a diagnostic tool for OT regarding sensitivity and specificity. Also, it has potential importance in the interpretation and differential diagnosis of OT. Thus, serum anti-Toxocara IgG ELISA should be a routine test for screening of suspected cases.

The gullet worms, classical Gongylonema pulchrum and newly differentiated Gongylonema nepalensis, are prevalent in various mammals in Japan and Sardinia, Italy, respectively. The former species is cosmopolitan in distribution, dwelling in the mucosa of the upper digestive tract of a variety of domestic and wild mammals, and also humans. At present, the geographical distribution of G. nepalensis is known in Nepal and Sardinia, with the nematode having been recorded from the oesophagus of water buffaloes (Nepal), cattle, sheep, goats and wild mouflon (Sardinia). To clarify their natural transmission cycles among domestic and wild mammals, the present study analysed the ribosomal RNA gene (rDNA) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) of worms of various origins: G. pulchrum worms from sika deer, wild boars, Japanese macaques, and feral alien Reeves's muntjacs in Japan, and G. nepalensis worms from a red fox and a wild boar in Sardinia. Although the internal transcribed spacer (ITS) regions of rDNA and partial cox1 nucleotide sequences of G. pulchrum from native wild mammals in Japan were distinct from those of the worms in cattle, the worms from feral alien Reeves's muntjacs showed the cattle-type ITS genotype and cox1 cattle-I and II haplotypes. The rDNA and cox1 nucleotide sequences of G. nepalensis from a red fox in Sardinia were almost identical to those of the worms from domestic and wild ruminants on the island. The ecological interaction between domestic and wild mammals and their susceptibility to different Gongylonema spp. must be considered when trying to elucidate this spirurid's transmission dynamics in nature.

Four representatives of the genus Lecithaster and one representative of the genus Hysterolecithoides were found during investigation of the trematode fauna of fish species in Vietnamese, Japanese and eastern coastal waters of the Russian Far East. Based on morphometric data, adult trematodes from Vietnamese Strongylura strongylura and Russian Acanthogobius flavimanus were identified as Lecithaster confusus, trematodes from Vietnamese Hemirhamphus marginatus as L. sayori and from osmerid fishes as L. salmonis. Further, a single specimen of Lecithaster sp. and representatives of Hysterolecithoides epinepheli were found in Vietnamese Siganus fuscescens. Morphological and molecular data, including 18S ribosomal DNA (rDNA) V4 fragment, 28S rDNA D1-D3 fragment, internal transcribed spacers (ITS) and a mitochondrial COI gene fragment were analysed for Lecithaster spp. The results revealed that L. sayori and L. salmonis are not synonyms of L. stellatus and L. gibbosus, respectively, but that Hysterolecithoides frontilatus and H. guangdongensis are junior synonyms of H. epinepheli. The 28S-rDNA-based phylogenetic tree of Hemiuroidea showed a distinct position for the genus Lecithaster with internal differentiation into three subclades, including L. confusus, L. sayori and Lecithaster sp. within the first subclade, L. mugilis and L. sudzuhensis within the second subclade and L. salmonis and L. gibbosus within the third subclade. Bayesian phylogenetic reconstructions of Hemiuroidea showed four clades for members of Hemiuridae and Lecithasteridae. The first clade consisted of Hemiuridae representatives and the second clade represented the genus Lecithaster. The third clade included genera Aponurus and Lecithophyllum (Lecithasteridae) and the fourth clade combined members of lecithasterid Quadrifoliovariinae and Hysterolecithinae and hemiurid Opisthadeninae and Bunocotylidae with high statistical support.

A novel microfilarial sheath protein (MfP) of the human filarial parasite Wuchereria bancrofti and its proinflammatory activity on host macrophages were identified recently. MfP is a homolog of the nematode bestrophin-9 superfamily that acts as a ligand of macrophage Toll-like receptor 4 (TLR4) to induce inflammation through NF-κB activation. Therefore, the presence and functional implication of this novel protein in adult-stage parasites were open questions to answer. In this study, the bovine filarial parasite Setaria cervi was used to simulate adult W. bancrofti. We detected the presence of MfP in adult-stage S. cervi through clear immunological cross-reactivity and immunolocalization employing an anti-MfP antibody developed in mice. Therefore, our findings put forward S. cervi as a cost-effective source of immunodominant filarial antigen MfP to simulate its future utilization in the immunotherapeutic intervention of lymphatic filariasis.

A new species of Paurodontoides, P. siddiqii n. sp., is described and illustrated based on its morphological, morphometric, and molecular characters. The new species is characterized by a female 550–729 μm long, lip region continuous with body contour, stylet length 7.0–8.0 μm long or c. 1.0–1.2 times the lip region diameter, lateral fields with four smooth incisures, excretory pore at 85–125 μm from anterior end located at the base of the pharyngeal bulb or posterior to it, basal pharyngeal bulb with a short posterior extension projecting into the intestine, monodelphic–prodelphic reproductive system with prominent 19–22 μm long post-uterine sac, and elongate conoid tail with a filiform terminus. The new species is compared with two known species of the genus. It differs from the type species of the genus, P. linfordi, by having slightly shorter stylet, lateral field with smooth incisures, different position of the excretory pore, and absence of male. Compared to P. latus, the new species has a shorter body, shorter stylet, different position of the excretory pore, female tail shape and absence of male. The new species was also compared with close species of the genus Paurodontus because of lateral field marked with four lines, asymmetrical stylet knobs and absence of male. Molecular phylogenetic studies of the new species using partial sequences of 18S rDNA revealed that it forms a clade with a species of the genus Ficotylus. In phylogenetic analyses using partial sequences of the 28S rDNA D2-D3 domain, the new species formed a monophyletic group with a species of the genus Veleshkinema and Sphaerularia spp. (Sphaerulariinae).

The complex life cycle of Trichinella spiralis includes the migration of newborn larvae through the bloodstream to their encystment in muscle. The parasite establishes an intimate contact with the erythrocytes of the host both during the migration of the newborn larvae and when encysting, as this parasite causes intense vascularization in the muscle cell. The goal of this work was to study the effects of various concentrations of T. spiralis muscle larvae (ML) on erythrocyte membranes. The treatment was performed by incubating human erythrocytes with equal volume of different concentrations of ML for 30 minutes, with controlled agitation (37°C). The control erythrocytes (with no contact with the larvae) were incubated in the same way with an equal volume of physiological solution. To evaluate the alterations to the erythrocytes by the action of the larvae and in the respective controls, an Erythrocyte Rheometer and a Digital Image Analysis technique were used. The results indicated that when the larval concentration was higher, the aggregation and erythrocyte membrane alterations were also higher. Also, the erythrocyte deformability index and the erythrocyte elasticity increased. The values of isolated cell coefficient varied from 0.51 in the treatment with 100 larvae/ml to 0.91 in the incubation with 1000 larvae/ml. This experiment shows that T. spiralis muscle larvae affect significantly the red blood cell aggregation and the erythrocyte viscoelastic properties.

Members of the genus Neoechinorhynchus Stiles & Hassall, 1905 are endoparasites of freshwater fishes, brackish water fishes, and freshwater turtles distributed worldwide. In North America, 33 species have been described. One of the most widely distributed species in the eastern United States and Canada is Neoechinorhynchus (Neoechinorhynchus) cylindratus, a common acanthocephalan that infects centrarchid fishes. In the current study, adult specimens of N. (N) cylindratus were collected from largemouth bass (Micropterus salmoides) from the Purificación River in northern Mexico. In the same freshwater system, two additional congeneric species (Neoechinorhynchus (Neoechinorhynchus) emyditoides and Neoechinorhynchus (Neoechinorhynchus) panucensis) were collected and analysed. Sequences of the large subunit, internal transcribed spacers ITS1 and ITS2, 5.8S from nuclear DNA, and sequences of the cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA were generated and aligned with other sequences obtained from GenBank. Maximum likelihood and Bayesian inference analyses inferred for each dataset showed that N. (N) panucensis, N. (N) emyditoides and N. (N) cylindratus were nested within several clades, indicating that these species do not share a common ancestor. Our phylogenies also revealed that the genus Neoechinorhynchus is paraphyletic, requiring further taxonomic revision using phylogenetic systematics and re-examination of morphological and ecological data. The presence of several N. (N) cylindratus adults in northern Mexico allowed us to typify this species for the first time using a combination of morphological and molecular characteristics. The current record shows a wide distribution range of N. (N) cylindratus across Canada, the United States and Mexico in the Nearctic region.

The genus Megalobatrachonema is a rare group of nematode parasites within Ascaridida. The systematic status of Megalobatrachonema in the superfamily Cosmocercoidea (Ascaridida) has long been controversial. The relationship of Megalobatrachonema and Chabaudgolvania remains unsolved. In the present study, a new species of Megalobatrachonema, M. hainanensis sp. nov., was described based on specimens collected in Amolops hainanensis (Boulenger) and Hylarana spinulosa (Smith) (Amphibia: Anura) from Hainan Island, China. The large ribosomal DNA (28S) and internal transcribed spacer (ITS1-5.8S-ITS2) were also sequenced for molecular identification and phylogenetic study. Phylogenetic analyses using maximum likelihood (ML) inference and Bayesian inference (BI) based on 28S and ITS1 sequence data, respectively, supported that Megalobatrachonema is a member of the family Kathlaniidae. In addition, the genetic comparison and phylogenetic results based on ITS1 sequence data also supported that the genus Chabaudgolvania should be considered as a synonym of Megalobatrachonema.

Species identification based on the morphometry of opisthaptoral hard parts, in combination with internal transcribed spacer ribosomal DNA (ITS rDNA) region sequences, confirmed the presence of four viviparous Gyrodactylus von Nordman, 1832 (Plathyhelminthes, Monogenea) species on Nototheniid fish from the Prince Gustav Channel (Weddell Sea, Antarctica). Gyrodactylus antarcticus Gusev, 1967 was found mostly on Trematomus newnesi Boulenger (93 specimens) but also on T. bernacchii Boulenger (one specimen), the latter representing a new host record for this species. Gyrodactylus byrdi Hargis & Dillon, 1968 and G. coriicepsi Rokicka, Lumme & Ziętara, 2009 were recorded on their type hosts, T. newnesi and Notothenia coriiceps Richardson, respectively. Gyrodactylus wilkesi Hargis & Dillon, 1968 was found mostly on the fins of T. bernacchii (29 specimens), but also on T. hansoni Boulenger (one specimen) and T. newnesi (three specimens). The finding of G. wilkesi on T. newnesi represents a new host record. The low number of Gyrodactylus specimens may indicate an accidental infection. The occurence of all four Gyrodactylus species in the Prince Gustav Channel represents a new locality record. According to phylogentic methods, the newly redescribed monogeneans belong to the Antarctic lineage, forming a sister group to North American and European marine Gyrodactylus species, and consist of two species groups, one comprising G. coriicepsi and G. nudifronsi Rokicka, Lumme & Ziętara, 2009, and the other G. anarcticus and G. wilkesi.