Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces.

BACKGROUND: Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques. RESULTS: Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation. CONCLUSION: The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.

Ecto- and endoparasites of the King's skink (Egernia kingii) on Penguin Island.

Wildlife species are often host to a diversity of parasites, but our knowledge of their diversity and ecology is extremely limited, especially for reptiles. Little is known about the host-parasite ecology of the Australian lizard, the King's skink (Egernia kingii). In spring of 2015, we carried out a field-based study of a population of King's skinks on Penguin Island (Western Australia). We documented five species of parasites, including two ectoparasitic mites (an undescribed laelapid mite and Mesolaelaps australiensis), an undescribed coccidia species, and two nematode species (Pharyngodon tiliquae and Capillaria sp.). The laelapid mite was the most abundant parasite, infesting 46.9% of the 113 captured lizards. This mite species increased in prevalence and abundance over the course of the study. Infection patterns of both mites varied with lizard life-stage; sub-adults were more commonly infested with laelapid mites than adults or juveniles, and sub-adults and adults were infested by more laelapid mites than juveniles. By contrast, adults had a higher prevalence of M. australiensis than juveniles or sub-adults. Among the gastrointestinal parasites, P. tiliquae was relatively common among the sampled lizards (35.3%). These results give new important information about reptiles as parasite hosts and what factors influence infection patterns.

Effective eradication of pinworms (Syphacia obvelata and Aspiculuris tetraptera) with Polygonum cognatum Meissn.

Polygonum cognatum Meissn. (Polygonaceae) is a wild edible plant known locally as "solucanotu, madimak" in Turkey and it has been used for various purposes, such as diuretic and antidiabetic, as well to treat oxyuris and worms internally in humans. The present study was carried out to investigate the anthelmintic activity of various extracts were obtained from the aerial parts of P. cognatum in order to justify its traditional usage in Turkish folk medicine. n-Hexane, ethyl acetate and methanol extracts were prepared from the aerial parts of the plant, successively. The anthelmintic potentials of these extracts were evaluated on mice naturally infected with Syphacia obvelata and Aspiculuris tetraptera. Cellophane perianal tape test and fecal flotation test were used for the activity screening. The results were compared with control and reference groups. In addition to all these experiments, the total amount of phenolic compounds and tannin contents thought to be responsible for the activity were evaluated for all extracts. According to the results, it has been found that the methanolic extract reduced the number of S. obvelata (66.8%) and A. tetraptera (73.4%) eggs. Moreover, the methanolic extract include higher amount of total phenolic compound (48.75 ±â€¯0.82 mg of GA/g of extract) and tannin (30.04 ±â€¯0.22 mg TA/g of extract) than n-Hexane (17.21 ±â€¯0.75 mg of GA/g of extract; 6.82 ±â€¯0.41 mg TA/g of extract) and ethyl acetate (25.06 ±â€¯0.94 mg of GA/g of extract; 10.29 ±â€¯0.34 mg TA/g of extract); therefore the anthelmintic activity could be related to these constituents. The present study explored the anthelmintic potential of P. cognatum. Further studies should be conducted into the mechanism of these compounds' against helminths. This study can be an important step in the discovery of new anthelmintic agents.

Parasitic nematodes of the genus Syphacia Seurat, 1916 infecting Muridae in the British Isles, and the peculiar case of Syphacia frederici.

Syphacia stroma (von Linstow, 1884) Morgan, 1932 and Syphacia frederici Roman, 1945 are oxyurid nematodes that parasitize two murid rodents, Apodemus sylvaticus and Apodemus flavicollis, on the European mainland. Only S. stroma has been recorded previously in Apodemus spp. from the British Isles. Despite the paucity of earlier reports, we identified S. frederici in four disparate British sites, two in Nottinghamshire, one each in Berkshire and Anglesey, Wales. Identification was based on their site in the host (caecum and not small intestine), on key morphological criteria that differentiate this species from S. stroma (in particular the tail of female worms) and by sequencing two genetic loci (cytochrome C oxidase 1 gene and a section of ribosomal DNA). Sequences derived from both genetic loci of putative British S. frederici isolates formed a tight clade with sequences from continental worms known to be S. frederici, clearly distinguishing these isolates from S. stroma which formed a tight clade of its own, distinct from clades representative of Syphacia obvelata from Mus and S. muris from Rattus. The data in this paper therefore constitute the first record of S. frederici from British wood mice, and confirm the status of this species as distinct from both S. obvelata and S. stroma.

Helminth parasites of Mabuya arajara Rebouças-Spieker, 1981 (Lacertilia: Mabuyidae) from Chapada do Araripe, northeastern Brazil.

We provide data on the helminth fauna from the digestive tract of the lizard Mabuya arajara Rebouças-Spieker, 1981 from Chapada do Araripe, northeastern Brazil. Seventy one of the 127 lizards examined (56%) were infected with four nematode species: Physalopteroides venancioi and Physaloptera sp. (Physalopteridae), Strongyluris oscari (Heterakidae), and Parapharyngodon alvarengai (Pharyngodonidae), the latter being the component species (prevalence 53.5%; mean intensity of infection 3.37 ± 2.0; discrepancy index D = 0.69). The helminth P. alvarengai infected M. arajara throughout the year and showed increased infection rates in July, at the beginning of the dry season. In addition to the relationship with seasonality, lizards with greater body length and/or body mass were more infected. Relationships between number of parasites and body mass and with the sexes of lizards, on the other hand, were not found. Mabuya arajara represents a new host for these nematodes. This study contributes to the knowledge of the helminth fauna associated with the digestive tract of lizards from South America and the Caatinga domain.

Molecular characterization of cosmopolitan and potentially co-invasive helminths of commensal, murid rodents in Gauteng Province, South Africa.

Concurrent studies of helminth parasites of introduced and native rodent species are few and miss the opportunity to identify potential co-invasive parasite species. This study employed molecular tools to infer the phylogeny and elucidate the origin of potentially co-invasive parasites of commensal, murid rodents by assessing introduced Rattus norvegicus, Rattus rattus, Rattus tanezumi, and native Mastomys coucha in Gauteng Province, South Africa. Genotypes of Nippostrongylus brasiliensis recovered from R. norvegicus are nearly identical to those recovered from elsewhere in the world. The pinworms, Aspiculurus tetraptera, recovered from introduced R. tanezumi and R. rattus, Syphacia muris recovered from R. tanezumi, and Syphacia obvelata recovered from indigenous M. coucha have affiliations to those recovered of laboratory rodents from the USA and China. Syphacia obvelata was previously only known as a commensal endoparasite of laboratory rodents, and the S. muris genotype recovered from R. tanezumi in this study shows an affiliation to a genotype recovered from the same host species in Indonesia which is part of the native range. The study emphasizes the need for surveillance of potential co-invasive species and contributes in documenting genetic diversity of endoparasites of well-known hosts.

The diversity and evolution of nematodes (Pharyngodonidae) infecting New Zealand lizards.

Host-parasite co-evolutionary studies can shed light on diversity and the processes that shape it. Molecular methods have proven to be an indispensable tool in this task, often uncovering unseen diversity. This study used two nuclear markers (18S rRNA and 28S rRNA) and one mitochondrial (cytochrome oxidase subunit I) marker to investigate the diversity of nematodes of the family Pharyngodonidae parasitizing New Zealand (NZ) lizards (lygosomine skinks and diplodactylid geckos) and to explore their co-evolutionary history. A Bayesian approach was used to infer phylogenetic relationships of the parasitic nematodes. Analyses revealed that nematodes parasitizing skinks, currently classified as Skrjabinodon, are more closely related to Spauligodon than to Skrjabinodon infecting NZ geckos. Genetic analyses also uncovered previously undetected diversity within NZ gecko nematodes and provided evidence for several provisionally cryptic species. We also examined the level of host-parasite phylogenetic congruence using a global-fit approach. Significant congruence was detected between gecko-Skrjabinodon phylogenies, but our results indicated that strict co-speciation is not the main co-evolutionary process shaping the associations between NZ skinks and geckos and their parasitic nematodes. However, further sampling is required to fully resolve co-phylogenetic patterns of diversification in this host-parasite system.

Survey of intestinal helminths collected from pet rodents in México.

In this survey, intestinal helminths from pet rodents in Mérida, México, were analyzed. A total of 46 mice Mus musculus, 28 hamsters Mesocricetus auratus, 23 rats Rattus norvegicus, and 1 gerbil Meriones unguiculatus were purchased from six pet shops and one black market for wildlife in the city of Mérida. The overall prevalence of helminths in rodents was 61.2% (60/98). Six species of helminths were identified: the zoonotic cestode Rodentolepis nana, and the nematodes Aspiculuris tetraptera, Dentostomella translucida, Syphacia obvelata, Syphacia mesocriceti, and Syphacia muris. Of the 60 infected rodents, 25 (41.7%) harbored 2 or 3 species of helminths. Rodentolepis nana was found in 4.3% of mice and 17.9% of hamsters. This is the first report of infection with S. muris in pet rats. Considering the close physical contact between pet rodents and humans, the presence of R. nana in pets represents a potential risk of transmission, especially to children and immunocompromised individuals.

Parapharyngodon hugoi n. sp., a new nematode (Oxyuroidea: Pharyngodonidae) of the tree frog Trachycephalus typhonius (Linnaeus) from the Brazilian Pantanal, including a key to the congeners from amphibians of the American continent.

A new nematode Parapharyngodon hugoi n. sp. (Oxyuroidea: Pharyngodonidae) is described parasitising the large intestine of the tree frog Trachycephalus typhonius (Linnaeus) (Anura: Hylidae) from the wetlands of Pantanal, State of Mato Grosso do Sul, Brazil. The new species exhibits a unique structure of the posterior cloacal lip in males, which is supported by a rigid V-shaped structure. Parapharyngodon hylidae parasitic in hylid frogs, including T. typhonius, from Mexico, is the most similar congener to P. hugoi n. sp. but is distinguished from the new species by the presence of a gubernaculum (vs absence), by the lateral alae in males ending far anterior to cloacal opening (vs near to it) and because in gravid females the ovaries encircle the oesophageal corpus. Additionally, the new species differs from its congeners as well as from species of Thelandros Wedl, 1862, a very closely related genus, by the combination of features such as spicule length, number of caudal papillae, morphology of the anterior cloacal lip, which is echinate, and position of ovaries. The geographical distribution of hosts seems to play an important role in the speciation process of Parapharyngodon spp.; however, due the lack of molecular data this issue along with the validity of both Thelandros and Parapharyngodon are still questions to be solved in the future, after improvement of the genetic database. A key to the species of Parapharyngodon parasitic in amphibians from the American continent is provided.

Syphacia obvelata (Nematode, Oxyuridae) infecting laboratory mice Mus musculus (Rodentia, Muridae): phylogeny and host-parasite relationship.

Syphacia obvelata is a pinworm nematode parasite infecting man and laboratory animals in high abundance. This parasitological study was carried out during the period of March 2014-February 2015 to investigate the helminth parasites infecting the laboratory mice Mus musculus in the Animal House at Cairo University, Egypt. The prevalence of S. obvelata in M. musculus was 75.0 %. The extent of infection with S. obvelata is analyzed according to the sex of the host mice. It was shown that the prevalence of male infection was greater than female worms. Morphological characterization revealed that the present Oxyurid species possesses a rounded cephalic end with less developed lips, esophagus divided into cylindrical corpus, and globular bulb supported internally with valvular apparatus; three mamelons are located at the ventral surface with a single chitinized spicule and a gubernaculum provided with an accessory hook in males, and ovijector apparatus opens ventrally by the vulva surrounded by protruded lips in female worms. Body of the male was 0.623-1.130 (0.830 ± 0.11) mm long and 0.092-0.130 (0.110 ± 0.01) mm wide; the esophagus was 0.164-0.280 (0.210 ± 0.01) mm long; the nerve ring and excretory pore are located at 0.035-0.132 (0.073 ± 0.01) and 0.087-0.191 (0.145 ± 0.01) mm from the anterior end, respectively, while the female measured 2.930-4.650 (3.540 ± 0.1) mm long and 0.120-0.232 (0.156 ± 0.001) mm wide; the esophagus was 0.213-0.410 (0.342 ± 0.01) mm long; the nerve ring, excretory pore, and vulval opening are located at 0.026-0.157 (0.121 ± 0.01), 0.134-0.243 (0.195 ± 0.01), and 0.323-0.632 (0.546 ± 0.11) mm from the anterior end, respectively; eggs measured 0.120-0.139 (0.129 ± 0.001) mm long and 0.030-0.052 (0.045 ± 0.001) mm wide. It compared morphometrically with other Syphacia species described previously and showed little differences in measurements. Molecular characterization based on small subunit ribosomal DNA (rDNA) was done to confirm the obtained morphological and morphometric results. A preliminary genetic comparison between SSU rDNA of the present parasite and other species of Oxyuridae places it as a putative sister taxon to other S. obvelata.

Diagnosis of the pinworm Syphacia muris in the Wistar rat Rattus norvegicus.

This study aimed to compare three qualitative parasitological methods for the diagnosis of Syphacia muris infection in 30 Wistar rats (Rattus norvegicus) infected naturally. Methods of spontaneous sedimentation (Hoffman, Pons and Janer, or HPJ) and spontaneous flotation (Willis) for faecal samples and a method of taping (Graham) were performed and compared. The Graham and Willis methods were more sensitive than the HPJ method (P< 0.05). The Graham method was able to detect S. muris eggs in 100% of the samples. Eggs were detected in 83% and 60% of the samples using the Willis and HPJ methods, respectively. Method choice is important for screening for parasites of rats kept under laboratory conditions, as accurate diagnosis helps prevent future environmental contamination and infection. We concluded that the Graham method was the most efficient of those tested in this study for detection of S. muris infection in rats. This method is also rapid, inexpensive and practical, and should be implemented as a necessary measure for infection control.

Morphology of the oxyurid nematodes Trypanoxyuris (T.) cacajao n. sp. and T. (T.) ucayalii n. sp. from the red uakari monkey Cacajao calvus ucayalii in the Peruvian Amazon.

Cacajao calvus ucayalii (Thomas, 1928) (Primates: Pitheciidae), a subspecies endemic to the Peruvian Amazon, occurs in patchy and sometimes isolated populations in north-eastern Peru and is in a vulnerable situation, mainly due to habitat loss and hunting. This rareness and remote distribution means that, until now, parasitical studies have been limited. Based on optical and scanning electron microscopy of specimens of both sexes, we report two new species of Trypanoxyuris pinworms occurring in the large intestine of the Peruvian red uakari, namely Trypanoxyuris (Trypanoxyuris) cacajao and Trypanoxyuris (Trypanoxyuris) ucayalii. Both species showed a distinct morphology of the lips and cephalic structure. Sexual dimorphism in the lateral alae was observed in both male and the female worms, with ventral ornamentation being shown in the oesophageal teeth. The finding of these new pinworm species highlights the possibility of discovering other species.

Sequence variability in internal transcribed spacers of nuclear ribosomal DNA among isolates of the oxyurid nematodes Syphacia obvelata and Aspiculuris tetraptera from mice reared in laboratories in China.

This study examined sequence variability in internal transcribed spacers (ITS) of nuclear ribosomal DNA among Syphacia obvelata and Aspiculuris tetraptera isolates from laboratory mice from different geographical locations in China. ITS1, 5.8S and ITS2 rDNA were amplified separately from adult S. obvelata and A. tetraptera individuals by polymerase chain reaction (PCR), and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of ITS1, 5.8S and ITS2 rDNA from both nematodes were 314 bp and 456 bp, 157 bp, and 273 bp and 419 bp, respectively. The intraspecific sequence variations in S. obvelata ITS1 were 0-0.3%. For A. tetraptera they were 0-0.7% in ITS1 and 0-1.0% in ITS2. However, the interspecific sequence differences among members of the infraorder Oxyuridomorpha were significantly higher, being 54.0-65.5% for ITS1 and 55.3-64.1% for ITS2. Phylogenetic analysis based on the combined partial sequences of ITS1 and ITS2 using three inference methods - Bayesian inference, maximum likelihood and maximum parsimony - revealed that all the S. obvelata and A. tetraptera samples formed independent monophyletic groups. Syphacia obvelata was closer to Syphacia muris than to A. tetraptera, consistent with morphological classification. These results demonstrate that ITS1 and ITS2 rDNA sequences are useful markers for population genetic studies of oxyurid nematodes.

Bank voles (Myodes glareolus) and house mice (Mus musculus musculus; M. m. domesticus) in Europe are each parasitized by their own distinct species of Aspiculuris (Nematoda, Oxyurida).

The molecular phylogeny and morphology of the oxyuroid nematode genus Aspiculuris from voles and house mice has been examined. Worms collected from Myodes glareolus in Poland, Eire and the UK are identified as Aspiculuris tianjinensis, previously known only from China, while worms from Mus musculus from a range of locations in Europe and from laboratory mice, all conformed to the description of Aspiculuris tetraptera. Worms from voles and house mice are not closely related and are not derived from each other, with A. tianjinensis being most closely related to Aspiculuris dinniki from snow voles and to an isolate from Microtus longicaudus in the Nearctic. Both A. tianjinensis and A. tetraptera appear to represent recent radiations within their host groups; in voles, this radiation cannot be more than 2 million years old, while in commensal house mice it is likely to be less than 10,000 years old. The potential of Aspiculuris spp. as markers of host evolution is highlighted.

Two new species of Syphacia (Nematoda: Oxyuridae) in endemic murid rodents from Sulawesi, Indonesia.

Two new species of Syphacia (Nematoda: Oxyuridae) are described from endemic murids of Sulawesi, Indonesia: Syphacia (Syphacia) taeromyos sp. n. and S. (S.) paruromyos sp. n. parasitic in the caecum of Taeromys celebensis and Paruromys dominator, respectively. They are readily distinguished from all of the congeners recorded from Indonesia-Australian regions by having a round cephalic plate, vesicular lateral alae in the male, posteriorly positioned excretory pore in the male, and/or lacking cervical alae. Syphacia (S.) paruromyos differs from S. (S.) taeromyos by having a whip-like tail appendage in the male, longer relative distance between excretory pore and vulva, and larger eggs. The round cephalic plate in both sexes and developed vesicular lateral alae in the male are morphological traits common in endemic Syphacia species hitherto known from Sulawesi murids, suggesting that they have derived from a common ancestor and evolved with their hosts in the isolated insular environment.

Syphacia sp. (Nematoda: Oxyuridae) in coprolites of Kerodon rupestris Wied, 1820 (Rodentia: Caviidae) from 5,300 years BP in northeastern Brazil.

We present the results of paleoparasitological analyses in coprolites of Kerodon rupestris, rodent endemic to rocky areas of Brazil's semiarid region. The coprolites were collected from excavations at the archaeological site of Toca dos Coqueiros, in the National Park of Serra da Capivara, southeastern of state of Piauí. Syphacia sp. (Nematoda: Oxyuridae) eggs were identified in coprolites dated at 5,300 ± 50 years before present. This is the first record of the genus Syphacia in rodent coprolites in the Americas.

A phylogenetic assessment of the colonisation patterns in Spauligodon atlanticus Astasio-Arbiza et al., 1987 (Nematoda: Oxyurida: Pharyngodonidae), a parasite of lizards of the genus Gallotia Boulenger: no simple answers.

Parasite taxonomy traditionally relies on morphometric and life-cycle characteristics which may not reflect complex phylogenetic relationships. However, genetic analyses can reveal cryptic species within morphologically described parasite taxa. We analysed the phylogenetic variation within the nematode Spauligodon atlanticus Astasio-Arbiza, Zapatero-Ramos, Ojeda-Rosas & Solera-Puertas, 1987, a parasite of the Canarian lizard genus Gallotia Boulenger, inferring the origin of their current association. We also attempted to determine its relationship with other Spauligodon spp. Three different markers, mitochondrial COI plus nuclear 18S and 28S ribosomal RNA, were used to estimate the evolutionary relationships between these nematodes. S. atlanticus was found to be paraphyletic, suggesting that Gallotia spp. were colonised by two independent lineages of Spauligodon. Additional analyses of other Spauligodon spp. are required for a more complete interpretation of the evolution of this genus from the Canarian archipelago and its closest taxa. Our results emphasise the importance of extensive sampling and phylogenetic studies at the intrageneric level, and highlight the limitations of a morphologically based taxonomy in these parasites.

Redescription of Syphacia venteli Travassos 1937 (Nematoda: Oxyuridae) from Nectomys squamipes in Argentina and Brazil and description of a new species of Syphacia from Melanomys caliginosus in Colombia.

Syphacia venteli Travassos, Mem Inst Oswaldo Cruz 32:607-613, 1937 is redescribed on the basis of specimens recovered from the type host, Nectomys squamipes (Brants 1827), from Brazil and Argentina. Specimens determined by Quentin (Bull Mus Natl Hist Nat 2:909-925, 1969) as S. venteli from Melanomys caliginosus (Tomes 1860) in Colombia were re-studied and assigned to a new species. In both species, structures such as the shape of the cephalic plate, details and distribution of the submedian papillae and amphids, presence and absence of the lateral and cervical alae, and shape and structure of the accessory hook of the gubernaculum were studied with the light microscope and scanning electron microscope. The present survey is the first detailed study of the species S. venteli since the original description, and the first record of this species from Argentina. Moreover, the present study suggests that the validity of some host species of Syphacia should be questioned and that through of the study of deposited specimens, a more exact number of parasitized host species can be confirmed, contributing to a better understanding of host specificity in this genus.

Description of Lemuricola (Lemuricola) pongoi--male (Nematoda: Enterobiinae) parasitising orangutan Pongo abelii.

The study presents the first description of male specimen (allotype) of the pinworm species Lemuricola (Lemuricola) pongoi, parasitising Sumatran orangutan (Pongo abelii) from Sumatra (Indonesia). The male specimen morphology presents all features fully corresponding with diagnose of the genus and subgenus Lemuricola. We are agreeing that Protenterobius is synonymous with Lemuricola because the length of tail appendix in males is not a sufficient feature for separation of the genera or subgenera. For L. (L.) pongoi male are characteristic features: body length (4,803 microm), total oesophagus length (530), tail length (235), tip of tail length (188) and spicule length (90).

Oxyurid nematodes of pet rodents in Slovakia - a neglected zoonotic threat.

The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.