Prevalence of Soil-Transmitted Helminths and Molecular Clarification of Hookworm Species in Ethnic Ede Primary Schoolchildren in Dak Lak Province, Southern Vietnam.

To know the infection status of helminths in primary schoolchildren of southern parts of Vietnam, we performed an epidemiological study in Krong Pac district, Dak Lak Province, Vietnam. A total of 1,206 stool specimens were collected from ethnic Ede schoolchildren in 4 primary schools in 2015 and examined by the Kato-Katz technique. In addition, stool cultures were done by the Harada-Mori method to obtain hookworm larvae and then to clarify the species of hookworms infected. The results showed that the helminth infection rate was 25.0%, including 2.0% Ascaris lumbricoides, 0.33% Trichuris trichiura, and 22.8% hookworm infections. The average intensity of infection was 102.0 eggs per gram of feces (EPG) for Ascaris, 36.0 EPG for Trichuris, and 218.0 EPG for hookworms. ITS1 gene sequences of the hookworm larvae were identical with those of Necator americanus (100% homology) reported in GenBank. It has been confirmed in this study that the hookworm, N. americanus, is a dominant helminth species infected in primary schoolchildren of a southern part of Vietnam. Public health attention is needed for control of hookworm infections among schoolchildren in surveyed areas of Vietnam.

Identification and Prevalence of Globocephalus samoensis (Nematoda: Ancylostomatidae) among Wild Boars (Sus scrofa coreanus) from Southwestern Regions of Korea.

This study describes the first record of Globocephalus samoensis (Nematoda: Ancylostomatidae) recovered in wild boars from southwestern regions of Korea. Gastrointestinal tracts of 111 Korean wild boars (Sus scrofa coreanus) hunted from mountains in Suncheon-si, Gwangyang-si, and Boseong-gun between 2009 and 2012 were examined for their visceral helminths. G. samoensis, as identified by morphological characteristics of the head and tail, were recovered from the small intestine of 51 (45.9%) wild boars. Worms were found from 7 of 28 wild boars (25.0%) from Suncheon-si, 40 of 79 (50.6%) from Gwangyang-si, and all 4 (100%) from Boseong-gun. The length of adult females was 7.2±0.5 mm, and the thickest part of the body measured the average 0.47±0.03 mm, while those of males were 6.52±0.19 and 0.37±0.02 mm, respectively. The buccal cavity was equipped with a pair of large and bicuspid subventral lancets near the base of the capsule. The average length of spicules of males was 0.45±0.02 mm. By the present study, G. samoensis is recorded for the first time in southwestern regions of Korea. Additionally, morphological characteristics and identification keys provided in the present study will be helpful in the faunistic and taxonomic studies for strongylid nematodes in both domestic and wild pigs. The infection of G. samoensis apparently did not elicit pathologic lesions, as revealed by macroscopic observation during the autopsy of all wild boars in this study.

Morphometric and molecular characterization of the species of Uncinaria Frölich, 1789 (Nematoda) parasitic in the Australian fur seal Arctocephalus pusillus doriferus (Schreber), with notes on hookworms in three other pinniped hosts.

This study presents morphological and molecular data on hookworms from the Australian fur seal Arctocephalus pusillus doriferus (Schreber) currently identified in Australian waters as Uncinaria hamiltoni Baylis, 1933. Additional specimens from the Australian sea lion Neophoca cinerea (Péron) and the New Zealand fur seal Arctocephalus forsteri (Lesson) from Australia, and the Southern elephant seal Mirounga leonina (Linnaeus) from Antarctica, were included. Using the internal transcribed spacer (ITS), hookworms from A. p. doriferus, N. cinerea and A. forsteri were found to be genetically similar but distinct from Uncinaria spp. found in M. leonina from Antarctica, as well as from Zalophus californianus (Lesson) and Callorhinus ursinus (Linnaeus) from California. Few morphological differences were detected between these taxa.

Arthrostoma leucurus sp. n. (Nematoda: Ancylostomatidae), A New Hookworm Species Isolated from Asian Badger in China.

PURPOSE: To date, ten validated Arthrostoma species were reported. Here, a new hookworm species was found from Asian badger (Meles leucurus). METHODS: Nineteen hookworms (9 males and 10 females) were collected from the small intestine of two Asian badgers in Xinjiang Uygur Autonomous Region, northwestern China. The hookworms were morphologically examined according to key taxonomic characters, such as anterior extremity direction, structures of oral opening (cutting plates or teeth), vulva location, buccal capsule anatomy (integrated or formed by articulating plates), the length of spicule and gubernaculum, number of plates of buccal capsule, and presence or absence of vulvar papillae. RESULTS: The hookworm species from Asian badger, here named as Arthrostoma leucurus sp. n., was different from the previously described ten Arthrostoma species. The phylogenetic tree based on the cox1 gene showed that Arthrostoma leucurus sp. n. formed a separate clade, as a sister group to Ancylostoma and Uncinaria species. CONCLUSION: Arthrostoma leucurus sp. n., the eleven validated Arthrostoma species, was identified from Asian badger in China.

[Molecular and morphological comparison of hookworms from genus Uncinaria invading red fox (Vulpes vulpes) and dog (Canis familiaris)]. / Morfologiczne i molekularne porównanie nicieni z rodzaju Uncinaria pasozytujacych u lisa (Vulpes vulpes) i psa (Canis familiaris).

Two species of hookworms from genus Uncinaria have been found so far in Poland. Uncinaria stenocephala infects mainly dog, wolf and red fox, whereas Uncinaria criniformis is a parasite of mustelids (but it was also reported from red fox). 19 male and 29 female hookworms from red foxes have been compared with 10 male and 12 female worms from dogs. Hookworms from dogs were generally smaller than these from foxes, but no other morphological differences could be found. These hookworms were qualified to species Uncinaria stenocephala on the ground of morphology of male. Genomic DNA samples have been isolated from these hookworms and segments of rDNA including part of small subunit of ribosomal RNA gene; internal transcribed spacer 1 (ITS1); 5.8 S ribosomal RNA; internal transcribed spacer 2 (ITS2) and part of large subunit of ribosomal RNA have been amplified and sequenced. Sequences from Uncinaria obtained both from foxes and dogs have shown very high similarity to the sequence of Uncinaria stenocephala, so all examined hookworms have been classified as belonging to this species.

Morphological variability and molecular identification of Uncinaria spp. (Nematoda: Ancylostomatidae) from grizzly and black bears: new species or phenotypic plasticity?

The hookworms Uncinaria rauschi Olsen, 1968 and Uncinaria yukonensis ( Wolfgang, 1956 ) were formally described from grizzly ( Ursus arctos horribilis) and black bears ( Ursus americanus ) of North America. We analyzed the intestinal tracts of 4 grizzly and 9 black bears from Alberta and British Columbia, Canada and isolated Uncinaria specimens with anatomical traits never previously documented. We applied morphological and molecular techniques to investigate the taxonomy and phylogeny of these Uncinaria parasites. The morphological analysis supported polymorphism at the vulvar region for females of both U. rauschi and U. yukonensis. The hypothesis of morphological plasticity for U. rauschi and U. yukonensis was confirmed by genetic analysis of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. Two distinct genotypes were identified, differing at 5 fixed sites for ITS-1 (432 base pairs [bp]) and 7 for ITS-2 (274 bp). Morphometric data for U. rauschi revealed host-related size differences: adult U. rauschi were significantly larger in black bears than in grizzly bears. Interpretation of these results, considering the historical biogeography of North American bears, suggests a relatively recent host-switching event of U. rauschi from black bears to grizzly bears which likely occurred after the end of the Wisconsin glaciation. Phylogenetic maximum parsimony (MP) and maximum likelihood (ML) analyses of the concatenated ITS-1 and ITS-2 datasets strongly supported monophyly of U. rauschi and U. yukonensis and their close relationship with Uncinaria stenocephala (Railliet, 1884), the latter a parasite primarily of canids and felids. Relationships among species within this group, although resolved by ML, were unsupported by MP and bootstrap resampling. The clade of U. rauschi, U. yukonensis, and U. stenocephala was recovered as sister to the clade represented by Uncinaria spp. from otariid pinnipeds. These results support the absence of strict host-parasite co-phylogeny for Uncinaria spp. and their carnivore hosts. Phylogenetic relationships among Uncinaria spp. provided a framework to develop the hypothesis of similar transmission patterns for the closely related U. rauschi, U. yukonensis, and U. stenocephala.

Arthrostoma miyazakiense (Nagayosi 1955) Comb. n., a parasite of the raccoon-like dog, Nyctereutes procyonoides, with a key to the genus Arthrostoma (Nematoda: Ancylostomatidae).

Necator miyazakiensis Nagayosi 1955, a hookworm from the raccoon-like dog, Nyctereutes procyonoides, in Miyazaki Prefecture, Kyushu, Japan, is redescribed and placed in the genus Arthrostoma Cameron 1927, and compared with the 6 other species of the genus. Arthrostoma miyazakiense comb. n. is found in raccoon-like dogs not only in Miyazaki Prefecture, but also in Kyoto and Hokkaido Prefectures. This is the first record of a species of Arthrostoma in Japan.

Two new nematodes from the Iriomote cat, Prionailurus iriomotensis, from Okinawa: Uncinaria (Uncinaria) maya n. sp. (Ancylostomatoidea) and Molineus springsmithi yayeyamanus n. subsp. (Trichostrongyloidea).

Uncinaria (Uncinaria) maya n. sp. (Nematoda: Ancylostomatidae) and Molineus springsmithi yayeyamanus n. subsp. (Nematoda: Molineidae) are described from the Iriomote cat, Prionailurus iriomotensis, on Iriomote Island, Okinawa, Japan. Uncinaria (U.) maya resembles Uncinaria (Uncinaria) felidis Maplestone, 1939, from Prionailurus bengalensis of India but is distinguished in that the body is much smaller, the ventral rays are set closely with the lateral rays, and the externolateral ray is much shorter than other laterals. Molineus springsmithi yayeyamanus differs from Molineus springsmithi springsmithi Inglis and Ogden, 1965, from Prionailurus bengalensis horsfieldi of East Nepal in that the body is much longer, whereas the esophagus is somewhat shorter and the spicules are divided more distally. Presence of the closely related nematodes in both the Iriomote cat and P. bengalensis suggests a close evolutionary relationship of the 2 hosts.

Molecular and morphometric evidence for separate species of Uncinaria (Nematoda: Ancylostomatidae) in California sea lions and northern fur seals: hypothesis testing supplants verification.

California sea lions (Zalophus californianus) and northern fur seals (Callorhinus ursinus) are each believed to host distinct hookworm species (Uncinaria spp.). However, a recent morphometric analysis suggested that a single species parasitizes multiple pinniped hosts, and that the observed differences are host-induced. To explore the systematics of these hookworms and test these competing hypotheses, we obtained nucleotide sequences of nuclear ribosomal DNA (D2/D3 28S, D18/D19 28S, and internal transcribed spacer [ITS] regions) from 20 individual hookworms parasitizing California sea lion and northern fur seal pups where their breeding grounds are sympatric. Five individuals from an allopatric population of California sea lions were also sampled for ITS-1 and D18/D19 28S sequences. The 28S D2/D3 sequences showed no diagnostic differences among hookworms sampled from individual sea lions and fur seals, whereas the 28S D18/D19 sequences had one derived (apomorphic) character demarcating hookworms from northern fur seals. ITS sequences were variable for 7 characters, with 4 derived (apomorphic) states in ITS-1 demarcating hookworms from California sea lions. Multivariate analysis of morphometric data also revealed significant differences between nematodes representing these 2 host-associated lineages. These results indicate that these hookworms represent 2 species that are not distributed indiscriminately between these host species, but instead exhibit host fidelity, evolving independently with each respective host species. This evolutionary approach to analyzing sequence data for species delimitation is contrasted with similarity-based methods that have been applied to numerous diagnostic studies of nematode parasites.

Nematodes parasitizing domestic ruminants in Afghanistan.

A total of 12 nematode species were determined in the collection recovered from domestic ruminants in Afghanistan. Ten of them were found for the first time in this region: Trichocephalus globulosus, Trichostrongylus probolurus, Cooperia bisonis, Nematodirus spathiger, N. abnormalis, Thelazia rhodesi, Bunostomum phlebotomum, Haemonchus contortus, Dictyocaulus filaria and Oesophagostomum radiatum. Morphological and metrical characteristics of T. globulosus, T. skrjabini and C. bisonis, based on our own material, are added.

Scanning electron microscopy of hookworms. 2. Adult of arthrostoma longspiculum (Maplestone, 1931).

The surface structures of adult Arthrostoma longespiculum were studied with the aid of the scanning electron microscope. In the mouth opening, a pair of ventral cutting plates was present; each was semilunar, thick and rounded at the inner edge. Cervical papillae were paired, small and sharp. The vulvar papilla was a single, round, wart-like projection adjacent to the vulva.

Light microscopy and scanning electron microscopy of Bathmostomum sangeri Cobbold, 1879, of elephants.

Bathmostomum sangeri is an intestinal parasite of the elephant. Males measured 12.15-14.25 mm in length; females measured 14.98-17.68 mm in length. Buccal capsule is well-developed and funnel-shaped. There is a raised and transverse fissure ridge around the oral margin. The internal wall of the buccal capsule is raised into a series of circular ridges or lamellae. Teeth or cutting plates could not be seen. Spicules are stout, wing-like structures. The telamon is pear-shaped, but a gibernaculum is not present. There are two pairs of papillae on the either side of the cloacal opening. The female tail is gradually tepering.

Hypodontus macropi, Mönnig, 1929 from a Bennetts' wallaby Macropus rufogrisea.

Hypodontus macropi was found in Macropus rufogrisea. Males measured 12.73--13.93 mm in length; one female was 19.07 mm in length. The buccal capsule was funnel-shaped. The mouth opening was directed antero-ventrally. There was a pair of large cutting plates on the dorsal margin of the buccal capsule. The brusal rays were well-developed. Spicules were equal and each bore a cuticular wing. A gubernaculum and a telamon were present. The vulva was situated near the anus. The female tail, 0.163 mm in length, was suddenly tapering.

Molecular systematics of pinniped hookworms (Nematoda: Uncinaria): species delimitation, host associations and host-induced morphometric variation.

Hookworms of the genus Uncinaria have been widely reported from juvenile pinnipeds, however investigations of their systematics has been limited, with only two species described, Uncinaria lucasi from northern fur seals (Callorhinus ursinus) and Uncinaria hamiltoni from South American sea lions (Otaria flavescens). Hookworms were sampled from these hosts and seven additional species including Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus californianus), South American fur seals (Arctocephalus australis), Australian fur seals (Arctocephalus pusillus), New Zealand sea lions (Phocarctos hookeri), southern elephant seals (Mirounga leonina), and the Mediterranean monk seal (Monachus monachus). One hundred and thirteen individual hookworms, including an outgroup species, were sequenced for four genes representing two loci (nuclear ribosomal DNA and mitochondrial DNA). Phylogenetic analyses of these sequences recovered seven independent evolutionary lineages or species, including the described species and five undescribed species. The molecular evidence shows that U. lucasi parasitises both C. ursinus and E. jubatus, whereas U. hamiltoni parasitises O. flavescens and A. australis. The five undescribed hookworm species were each associated with single host species (Z. californianus, A. pusillus, P. hookeri, M. leonina and M. monachus). For parasites of otarids, patterns of Uncinaria host-sharing and phylogenetic relationships had a strong biogeographic component with separate clades of parasites from northern versus southern hemisphere hosts. Comparison of phylogenies for these hookworms and their hosts suggests that the association of U. lucasi with northern fur seals results from a host-switch from Steller sea lions. Morphometric data for U. lucasi shows marked host-associated size differences for both sexes, with U. lucasi individuals from E. jubatus significantly larger. This result suggests that adult growth of U. lucasi is reduced within the host species representing the more recent host-parasite association. Intraspecific host-induced size differences are inconsistent with the exclusive use of morphometrics to delimit and diagnose species of Uncinaria from pinnipeds.

New host and locality records of snake intestinal nematode Kalicephalus spp in Indonesia.

OBJECTIVE: To observe three species of Kalicephalus found in three species of snake (Ophiophagus hannah, Ptyas mucosus, and Naja Sputatrix) during research on Capture Snake for Trading in Java and Snake Biodiversity in Kalimantan Islands. METHODS: Specimens for light microscopy examination were fixed with warm 70% alcohol, cleared and mounted in lactophenol for wet mounting. Drawings were made with the aid of a drawing tube attached to a Nikon compound microscope. Measurements were given in micrometers (µ) as the average of findings, followed by the range in parentheses, unless otherwise stated. RESULTS: Kalicephalus (Costatus) indicus was found from 7 Ptyas mucosus, Kalicephalus bungari from 2 Naja sputatrix and 1 Kalicephalus (Costatus) indicus and Kalicephalus assimilis found from 1 Ophiophagus hannah. The morphology and measurement of three species of Kalicephalus found in this study were close to those described before. CONCLUSIONS: New finding of host of Kalicephalus (Costatus) indicus and Kalicephalus bungari was a snake species of Naja sputatrix. New records of locality were Kalimantan island as the new locality of Kalicephalus assimilis, and Java island was new locality of Kalicephalus (Costatus) indicus.

Structural restoration of nematodes and acanthocephalans fixed in high percentage alcohol using DESS solution and rehydration.

Ninety-five percent ethanol is the most widely used field and laboratory preservative for nematodes and other helminth specimens intended for use in molecular systematics. Preservation of nematodes in high-concentration alcohols results in structural dehydration artifacts, including shrinkage and body surface distortions sufficient to obscure features required for morphological identification and analysis, thereby compromising precise morphometrics. However, treating dehydrated nematodes using a solution of DMSO, disodium EDTA, and NaCl, followed by rehydration in water produces marked improvements in specimen shape and surface features, resulting from diffusion of water into the tissues and pseudocoelom as the internal salt concentration is reduced. Following rehydration, tissue samples can be obtained for molecular research and individuals can be fixed for morphological examination. This treatment method is demonstrated for species of 3 nematode genera that vary substantially in body size ( Baylisascaris , Uncinaria , and Bidigiticauda ). The method also works on nematodes that have been cut in half, provided the individuals are large enough to be folded and clamped during treatment. This method appears promising for other helminths, although for an acanthocephalan, the treatment restored the body surface but failed to reverse the retracted proboscis.