Morphology, genetic characterization and molecular phylogeny of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) from the endangered Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae).

BACKGROUND: The Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae) is an endangered species of mammal endemic to the Qinghai-Tibetan Plateau. Parasites and parasitic diseases are considered to be important threats in the conservation of the Tibetan antelope. However, our present knowledge of the composition of the parasites of the Tibetan antelope remains limited. METHODS: Large numbers of nematode parasites were collected from a dead Tibetan antelope. The morphology of these nematode specimens was observed using light and scanning electron microscopy. The nuclear and mitochondrial DNA sequences, i.e. small subunit ribosomal DNA (18S), large subunit ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1), were amplified and sequenced for molecular identification. Moreover, phylogenetic analyses were performed using maximum likelihood (ML) inference based on 28S and 18S + 28S + cox1 sequence data, respectively, in order to clarify the systematic status of these nematodes. RESULTS: Integrated morphological and genetic evidence reveals these nematode specimens to be a new species of pinworm Skrjabinema longicaudatum (Oxyurida: Oxyuridae). There was no intraspecific nucleotide variation between different individuals of S. longicaudatum n. sp. in the partial 18S, 28S, ITS and cox1 sequences. However, a high level of nucleotide divergence was revealed between the new species and its congeners in 28S (8.36%) and ITS (20.3-23.7%) regions, respectively. Molecular phylogenetic results suggest that the genus Skrjabinema should belong to the subfamily Oxyurinae (Oxyuroidea: Oxyuridae), instead of the subfamily Syphaciidae or Skrjabinemiinae in the traditional classification, as it formed a sister relationship to the genus Oxyuris. CONCLUSIONS: A new species of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) is described. Skrjabinema longicaudatum n. sp. represents the first species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope. Our results contribute to the knowledge of the species diversity of parasites from the Tibetan antelope, and clarify the systematic position of the genus Skrjabinema.

A new genus and species of pinworm (Nematoda, Oxyuridae) from the gray mouse opossum, Tlacuatzin canescens.

Tlacuatzoxyuris simpsoni n. gen. n. sp. is described from the cecum of the gray opossum, Tlacuatzin canescens, a species endemic to the deciduous dry forest of Mexico. The digestive tracts of four specimens were examined for parasites; three of these were archived in the American Museum of Natural History and one was a live capture. Relative to the other four monotypic genera of pinworms known to infect opossums, the new genus is diagnosed on the basis of a round cephalic plate with a semicircular stoma surrounded by a rim. In addition, males feature a prominent cephalic vesicle not fully developed in females, accounting for sexual dimorphism. The new species includes small worms that feature a conspicuous, not reticulated cephalic vesicle and semicircular stoma and lateral alae with two crests. In addition, the postcloacal cuticle of males features a small area with ornamentation between cloaca and submedial papillae. Finally, both spicule and gubernaculum are relatively short. Although the eggs of Tlacuatzoxyuris n. gen. are unknown, the conspicuous differences in traits used in the diagnosis of genera prompted us to propose a new genus for the new species. This is the first species of Oxyuridae reported in mouse opossums outside South America, and the fifth species of the family occurring in didelphimorph marsupials. This is an example of the usefulness of documenting the diversity of parasites associated with this unique clade of mammals through the examination of preserved tissues.

Intestinal parasitic infections and associated factors in children of three rural schools in Colombia. A cross-sectional study.

Rural children are one of the populations that are most vulnerable to gastrointestinal parasite infections. Such diseases decrease the quality of life and result in growth and cognitive delays in the long term. This cross-sectional study was conducted to determine the frequency of intestinal parasite infections among rural schoolchildren in the municipality of Apulo, Colombia. A total of 97 stool samples from children aged between 5 and 15 years were collected and examined via direct light microscopy. Microscopic examination was repeated with sediments obtained using a fecal parasite concentrator, and the Kato-Katz test was performed. Frequency of intestinal parasite infection was 100%. Endolimax nana (77.35%), Blastocystis sp. (71.1%), Giardia intestinalis (39.1%), Entamoeba coli (25.7%), and the Entamoeba histolytica/dispar/moshkovskii complex (9.2%) were the most prevalent protozoa. Trichuris trichiura was the most prevalent helminth (12.3%), followed by Enterobius vermicularis (6.15%) and Ascaris lumbricoides (5.1%). Among the analyzed associated factors, consumption of untreated water increased the risk of acquiring pathogenic intestinal parasites. Finally, because G. intestinalis was the most prevalent pathogenic protozoan, molecular analysis was conducted to establish genetic assemblages and subassemblages of Giardia through sequence-based genotyping of the glutamate dehydrogenase, triose phosphate isomerase, and beta-giardin genes. A total of 14 G. intestinalis-positive samples were genotyped, which revealed the presence of subassemblages AI (n = 1), AII (n = 7), BIII (n = 2), BIV (n = 2), and BIII/BIV (n = 1) as well as a mixed subassemblage AII + BIII (n = 1). Our results indicate that gastrointestinal parasite infections in the tested population were mainly caused by suboptimal water quality. Moreover, molecular typing of G. intestinalis suggested contamination of water by animal- and human-derived cysts.

Prevalence, Morphological and Molecular Phylogenetic Analyses of the Rabbit Pinworm, Passalurus ambiguus Rudolphi 1819, in the Domestic Rabbits Oryctolagus cuniculus.

INTRODUCTION: Passalurus ambiguus, a pinworm nematode parasite, infects domestic and wild rabbits, hares, and rodents worldwide. MATERIALS AND METHODS: The current parasitological study was performed during January-December 2016, to investigate helminth parasites infecting the domestic rabbit species Oryctolagus cuniculus at the Department of Animal Production, Faculty of Agriculture, Cairo University, Cairo, Egypt. RESULTS: Of the twenty rabbit specimens examined for gastrointestinal nematodes, 75% were infected with adult oxyurid species, which were morphologically characterized using light and scanning electron microscopy studies. The oxyurid species had a triangular mouth opening surrounded by simple lips with four cephalic papillae and a pair of lateral amphidial pores with three teeth-like structures, an esophagus divided into a cylindrical corpus and globular bulb supported internally with tri-radiate valvular apparatus, and four caudal papillae distributed on the posterior end of males with a single short protruding spicule and ovijector apparatus opening ventrally by the vulva, surrounded by protruded lips in female worms. The species were compared morphometrically with other Passalurus species described previously; light differences were found in different body part sizes. Molecular characterization based on 18 small subunit (SSU) rDNA sequences showed ~ 85% similarity with other Chromadorea species. A preliminary genetic comparison between the 18S rDNA sequences of the isolated parasite and those of other oxyurid species suggested that it belonged to Passalurus ambiguus. The 18S rDNA sequence of the parasite was deposited in GenBank (accession no., MG310151.1). CONCLUSION: The 18S rDNA gene of P. ambiguus was shown to yield a unique genetic sequence that confirms its taxonomic position within the Oxyuridae family.

Morphological and genetic characterization of Syphabulea tjanschani () (Nematoda: Oxyuridae), with phylogenetic position of Syphabulea in Oxyuridae.

Pinworms are important parasitic nematodes in animals and humans, and many species are of medical and veterinary importance. The genus Syphabulea is a poorly known group of pinworms. The systematic position of Syphabulea in Oxyuridae remains unclear. Moreover, there is still a paucity of detailed information on some morphological aspects of the type species S. tjanschani (Ablasov, 1962). In the present study, the detailed morphology of S. tjanschani was studied using light microscopy and, for the first time, scanning electron microscopy. The results revealed three traits useful for the characterization of the species including: cuticular depression around excretory pore, non-prominent labial teeth and number of adhesive ridges in three ventral mammelons. The ribosomal [small ribosomal DNA (18S), internal transcribed spacer (ITS) and large ribosomal DNA (28S)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions of S. tjanschani were also firstly amplified and sequenced for future use in the molecular identification of this poorly known species. In addition, in order to investigate the systematic position of Syphabulea in Oxyuridae, the phylogenetic analyses of representatives of the Oxyuridae were performed using maximum likelihood (ML) and maximum parsimony (MP) methods based on 18S, 28S and 18S + 28S + cox1 sequence data, respectively. The phylogenetic results based on different sequence data all supported the genus Syphabulea to be a member of the subfamily Syphaciinae. The phylogenetic analysis based on 28S sequence data also showed Syphabulea as sister to the genus Syphatineria. Our present study represents the first attempt to resolve the systematic position of the genus Syphabulea using phylogenetic analyses based on molecular data.

Genetic variation of Enterobius vermicularis among schoolchildren in Thailand.

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.

Pinworm diversity in free-ranging howler monkeys (Alouatta spp.) in Mexico: Morphological and molecular evidence for two new Trypanoxyuris species (Nematoda: Oxyuridae).

Two new species of Trypanoxyuris are described from the intestine of free-ranging howler monkeys in Mexico, Trypanoxyuris multilabiatus n. sp. from the mantled howler Alouatta palliata, and Trypanoxyuris pigrae n. sp. from the black howler Alouatta pigra. An integrative taxonomic approach is followed, where conspicuous morphological traits and phylogenetic trees based on DNA sequences are used to test the validity of the two new species. The mitochondrial cytochrome oxidase subunit 1 gene, and the nuclear ribosomal 18S and 28S rRNA genes were used for evolutionary analyses, with the concatenated dataset of all three genes used for maximum likelihood and Bayesian phylogenetic analyses. The two new species of pinworms from howler monkeys were morphologically distinct and formed reciprocally monophyletic lineages in molecular phylogenetic trees. The three species from howler monkeys, T. multilabiatus n. sp., T. pigrae n. sp., and Trypanoxyuris minutus, formed a monophyletic group with high bootstrap and posterior probability support values. Phylogenetic patterns inferred from sequence data support the hypothesis of a close evolutionary association between these primate hosts and their pinworm parasites. The results suggest that the diversity of pinworm parasites from Neotropical primates might be underestimated.

The complete mitochondrial genome of rabbit pinworm Passalurus ambiguus: genome characterization and phylogenetic analysis.

Passalurus ambiguus (Nematda: Oxyuridae) is a common pinworm which parasitizes in the caecum and colon of rabbits. Despite its significance as a pathogen, the epidemiology, genetics, systematics, and biology of this pinworm remain poorly understood. In the present study, we sequenced the complete mitochondrial (mt) genome of P. ambiguus. The circular mt genome is 14,023 bp in size and encodes of 36 genes, including 12 protein-coding, two ribosomal RNA, and 22 transfer RNA genes. The mt gene order of P. ambiguus is the same as that of Wellcomia siamensis, but distinct from that of Enterobius vermicularis. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference (BI) showed that P. ambiguus was more closely related to W. siamensis than to E. vermicularis. This mt genome provides novel genetic markers for studying the molecular epidemiology, population genetics, systematics of pinworm of animals and humans, and should have implications for the diagnosis, prevention, and control of passaluriasis in rabbits and other animals.

Sequence variability in four mitochondrial genes among pinworm Aspicularis tetraptera isolates from laboratory mice in four provinces, China.

This research aimed at exploring sequence variability in four mitochondrial (mt) genes, namely, cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb) and NADH dehydrogenase subunits 1 and 5 (nad1 and nad5), among pinworm Aspicularis tetraptera isolates from laboratory mice in four different provinces, China. A part of the cox1 (pcox1), cytb (pcytb), nad1 and nad5 genes (pnad1 and pnad5) were amplified separately from individual pinworms by polymerase chain reaction (PCR) and sequenced to determine sequence variations and examine their phylogenetic relationships. Herein, the intra-specific sequence variations within A. tetraptera were 0-0.5% for pcox1, 0-1.4% for pcytb, 0-1.8% for pnad1 and 0-1.7% for pnad5, respectively. In contrast, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 13.7-17.0% for pcox1, 24.5-34.7% for pcytb, 26.6-29.6% for pnad1 and 24.4-25.5% for pnad5, respectively. Three methods, namely, Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP), were used for phylogenetic analyses based on the combined sequences of the four mt gene sequences, and the results indicated that all A. tetraptera samples form monophyletic groups, but samples from the same geographical origin did not always cluster together. This study demonstrated the existence of low-level intra-specific variation in four mtDNA sequences among A. tetraptera isolates from laboratory mice in different geographic regions in China, indicating no obvious geographical distinction among A. tetraptera isolates in China. These findings have important implications for studying systematics, molecular epidemiology and population genetics of A. tetraptera.

Molecular paleoparasitological hybridization approach as effective tool for diagnosing human intestinal parasites from scarce archaeological remains.

Paleoparasitology is the science that uses parasitological techniques for diagnosing parasitic diseases in the past. Advances in molecular biology brought new insights into this field allowing the study of archaeological material. However, due to technical limitations a proper diagnosis and confirmation of the presence of parasites is not always possible, especially in scarce and degraded archaeological remains. In this study, we developed a Molecular Paleoparasitological Hybridization (MPH) approach using ancient DNA (aDNA) hybridization to confirm and complement paleoparasitological diagnosis. Eight molecular targets from four helminth parasites were included: Ascaris sp., Trichuris trichiura, Enterobius vermicularis, and Strongyloides stercoralis. The MPH analysis using 18th century human remains from Praça XV cemetery (CPXV), Rio de Janeiro, Brazil, revealed for the first time the presence E. vermicularis aDNA (50%) in archaeological sites of Brazil. Besides, the results confirmed T. trichiura and Ascaris sp. infections. The prevalence of infection by Ascaris sp. and E. vermicularis increased considerably when MPH was applied. However, a lower aDNA detection of T. trichiura (40%) was observed when compared to the diagnosis by paleoparasitological analysis (70%). Therefore, based on these data, we suggest a combination of Paleoparasitological and MPH approaches to verify the real panorama of intestinal parasite infection in human archeological samples.

Phylogenetic relationships between pinworms (Nematoda: Enterobiinae) parasitising the critically endangered orang-utan, according to the characterisation of molecular genomic and mitochondrial markers.

Pinworms (Nematoda: Enterobiinae) include 52 species parasitising primates throughout the world. In the present study, we performed the first ever molecular analysis to investigate the phylogenetic position of recently described pinworms parasitising the Sumatran orang-utan. The phylogenetic analysis of mitochondrial CO1 and chromosomal 18S rDNA and ITS1 regions could support the independent status of several Nematoda species. Our molecular data clearly suggest that Enterobius (Colobenterobius) buckleyi and Lemuricola (Protenterobius) pongoi together with Pongobius hugoti form separate clades among other studied species, which significantly supports the hypothesis of recently described new species parasitising the orang-utan (Pongo abelii and Pongo pygmaeus). The phylogenetic tree based on cytochrome oxidase 1 (CO1) gene variability showed possible close relationships between L. (Protenterobius) pongoi and P. hugoti; thus, we can assume that these species could have initially diverged in sympatry from a common ancestor.

Host specificity shapes population structure of pinworm parasites in Caribbean reptiles.

Host specificity is one of the potential factors affecting parasite diversification because gene flow may be facilitated or constrained by the number of host species that a parasite can exploit. We test this hypothesis using a costructure approach, comparing two sympatric pinworm parasites that differ in host specificity - Parapharyngodon cubensis and Spauligodon anolis - on the Puerto Rican Bank and St. Croix in the Caribbean. Spauligodon anolis specializes on Anolis lizards, whereas P. cubensis parasitizes Anolis lizards as well as many other species of lizards and snakes. We collected lizards from across the Puerto Rican Bank and St. Croix, sampled them for S. anolis and P. cubensis and generated nuclear and mitochondrial sequence data from the parasites. We used these data to show that P. cubensis is comprised of multiple cryptic species that exhibit limited population structure relative to S. anolis, which is consistent with our prediction based on their host specificity. We also provide evidence that the distribution of P. cubensis species is maintained by competitive exclusion, and in contrast to previous theoretical work, the parasites with the greatest number of host species also reach the highest prevalence rates. Overall, our results are consistent with the hypothesis that host specificity shapes parasite diversification, and suggest that even moderate differences in host specificity may contribute to substantial differences in diversification.

Genetic variation in mitochondrial DNA among Enterobius vermicularis in Denmark.

Despite being the most prevalent nematode infections of man in Western Europe and North America, our knowledge of the genetic variability in Enterobius vermicularis is fragmented. We here report on a genetic study of pinworms in Denmark, performed using the cytochrome oxidase I (cox1) gene, with DNA extracted from individual eggs collected from clinical (human) samples. We collected cellophane-tape-test samples positive for pinworm eggs from 14 Departments of Clinical Microbiology in Denmark and surface-sterilized the eggs using a 1% hypochlorite solution before performing conventional PCR. Twenty-two haplotypes were identified from a total of 58 Danish patients. Cluster analysis showed that all Danish worms grouped together with human samples from Germany and Greece and with samples from Japanese chimpanzees designated as 'type B'. Analysis of molecular variance showed no significant difference or trends in geographical distribution of the pinworms in Denmark, and several haplotypes were identical or closely related to samples collected in Germany, Greece and Japan. However, worms from the 4 countries were found to belong to different populations, with Fst values in the range of 0·16 to 0·47. This study shows pinworms in Denmark to be a homogenous population, when analysed using the cox1 mitochondrial gene.

Redescription of Enterobius (Enterobius) macaci Yen, 1973 (Nematoda: Oxyuridae: Enterobiinae) based on material collected from wild Japanese macaque, Macaca fuscata (Primates: Cercopithecidae).

Enterobius (Enterobius) macaci Yen, 1973 (Nematoda: Oxyuridae: Enterobiinae) was collected from a Japanese macaque, Macaca fuscata, in Nara and Yamaguchi Prefectures, Honshu Island, Japan, for the first time. A redescription is presented along with DNA sequence data. This pinworm is a typical member of the subgenus Enterobius and is characteristic in the spicule morphology, being readily distinguished from other congeners. Phylogenetic analyses based on 18S ribosomal RNA gene (rDNA) and mitochondrial DNA (mtDNA) Cox1 gene assign its position in the pinworm lineage adapted to the Old World primates, showing divergence before the splitting of the chimpanzee and human pinworms.

Phylogenetic relationships of rodent pinworms (genus Syphacia) in Japan inferred from 28S rDNA sequences.

The 28S rDNA from nine species of the genus Syphacia collected in Japan was sequenced, and the phylogenetic relationship was inferred from multiple sequence alignment of 28S rDNA by the MAFFT program. Phylogenetic tree indicates that S. petrusewiczi, which was the only species belonging to the subgenus Seuratoxyuris, has diverged earlier than other rodent pinworms examined and was distantly separated from the others genetically. It was revealed that S. agraria and S. vandenbrueli, whose subgeneric status has not been specified, belonged to the subgenus Syphacia together with other 6 species. Syphacia montana from Clethrionomys, Eothenomys and Microtus was very closely related to S. obvelata from Mus, and that S. frederici from Apodemus and S. vandenbrueli from Micromys were comparatively closely related to the former two species. The phylogenetic relationship among the three species of Syphacia found in Japanese Apodemus was inconsistent with the biogeography of host rodents. The co-evolutionary relationship between pinworm species and their host rodents may not be so strict and host switching has probably occurred frequently during the course of evolution.

Enterobiasis among preschool children: a study from Kayseri, Turkey.

The infection rate of Enterobius vermicularis was investigated in 1,070 preschool children aged 5-7 years in Kayseri, Turkey by cellotape anal swab from May-September 2005. The children's parents were asked to complete questionnaires inquiring into the potential risk factors involved. The overall egg positive rate for E. vermicularis was 5.1%. The infection rate among boys and girls was similar. The association between family size, household income/month, education level of the parents, employment status of the mother and enterobiasis were found to be significant.

Morphology of Enterobius (Colobenterobius) colobis Vuylstéke, 1964 (Nematoda: Oxyuridae: Enterobiinae) collected from an ashy red colobus, Procolobus rufomitratus tephrosceles (Elliot, 1907) (Primates: Cercopithecidae: Colobinae), in Uganda.

Enterobius (Colobenterobius) colobis Vuylstéke, 1964 (Nematoda: Oxyuridae) is redescribed based on males and females collected from an ashy red colobus, Procolobus rufomitratus tephrosceles (Elliot, 1907) (Primates: Cercopithecidae: Colobinae), in Uganda. Two morphotypes are recognized among females, which are readily distinguished by tail length, termination level of lateral alae, and egg size. The relative position of cellular wall greatly varied in the ovijector, indicating its limited systematic value. The males possess a much shorter spicule than those in previous descriptions, suggesting the presence of polymorphism.

Chimpanzee pinworm, Enterobius anthropopitheci (Nematoda: Oxyuridae), maintained for more than twenty years in captive chimpanzees in Japan.

The chimpanzee pinworm, Enterobius anthropopitheci (Gedoelst, 1916), was found in chimpanzees, Pan troglodytes, reared in Kumamoto Primate Research Park, Sanwa Kagaku Kenkyusho Co., Ltd., Kumamoto, Japan, in 2006. Because the chimpanzees in this institution originated from chimpanzees imported from Africa before 1984, it is considered that E. anthropopitheci infection has persisted for more than 20 yr in the chimpanzees. Analysis of pinworm specimens preserved in the institution revealed that transition of predominant pinworm species occurred, responding to the change of anthelmintics used for pinworm treatment. Present dominance of E. anthropopitheci is surmised to be caused by fenbendazole, which has been adopted from 2002. Scarcity of mixed infection with E. anthropopitheci and Enterobius vermicularis suggests interspecific competition between the pinworms.