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.

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.

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.

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.

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.

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.

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.

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) infection in raccoon dogs of Korea and experimental transmission to dogs.

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) is a hookworm species reported from the small intestines of raccoon dogs (Nyctereutes procyonoides) in Japan. Five Korean raccoon dogs (N. procyonoides koreensis) caught from 2002 to 2005 in Jeollanam-do (Province), a southeastern area of South Korea, contained helminth eggs belonging to 4 genera (roundworm, hookworm, whipworm, and Capillaria spp.) and cysts of Giardia sp. in their feces. Necropsy findings of 1 raccoon dog revealed a large number of adult hookworms in the duodenum. These hookworms were identified as Arthrostoma miyazakiense based on the 10 articulated plates observed in the buccal capsule and the presence of right-sided prevulval papillae. Eggs of A. miyazakiense were 60-65 x 35-40 micrometer (av. 62.5 x 35 micrometer), and were morphologically indistinguishable from those of Ancylostoma caninum. The eggs were cultured to infective 2nd stage larvae via charcoal culture, and 100 infective larvae were used to experimentally infect each of 3 mixed-bred puppies. All puppies harbored hookworm eggs in their feces on the 12th day after infection. This is the first report thus far concerning A. miyazakiense infections in raccoon dogs in Korea, and the first such report outside of Japan.

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) infection in raccoon dogs of Korea and experimental transmission to dogs

Arthrostoma miyazakiense (Nematoda: Ancylostomatidae) is a hookworm species reported from the small intestines of raccoon dogs (Nyctereutes procyonoides) in Japan. Five Korean raccoon dogs (N. procyonoides koreensis) caught from 2002 to 2005 in Jeollanam-do (Province), a southeastern area of South Korea, contained helminth eggs belonging to 4 genera (roundworm, hookworm, whipworm, and Capillaria spp.) and cysts of Giardia sp. in their feces. Necropsy findings of 1 raccoon dog revealed a large number of adult hookworms in the duodenum. These hookworms were identified as Arthrostoma miyazakiense based on the 10 articulated plates observed in the buccal capsule and the presence of right-sided prevulval papillae. Eggs of A. miyazakiense were 60-65 x 35-40 micrometer (av. 62.5 x 35 micrometer), and were morphologically indistinguishable from those of Ancylostoma caninum. The eggs were cultured to infective 2nd stage larvae via charcoal culture, and 100 infective larvae were used to experimentally infect each of 3 mixed-bred puppies. All puppies harbored hookworm eggs in their feces on the 12th day after infection. This is the first report thus far concerning A. miyazakiense infections in raccoon dogs in Korea, and the first such report outside of Japan.

First report and characterization of adult Uncinaria spp. in New Zealand Sea Lion (Phocarctos hookeri) pups from the Auckland Islands, New Zealand.

Two species of hookworms (Uncinaria lucasi and Uncinaria hamiltoni) have been formally described from pinnipeds, but dissimilar types are noted from these hosts. This report is the first description of hookworms (Uncinaria spp.) from the New Zealand sea lion, Phocarctos hookeri. The nematodes were collected from dead pups on Enderby Island (Auckland Islands, 50 degrees 30', 166 degrees 17') during January and February, 2004. Standard measurements of male and female hookworms were obtained, providing a general morphometric characterization of the hookworm species in P. hookeri. Considerable variations in the body length of adult hookworms were noted within the same host. The arrangement of some of the bursal rays differs from that described for U. lucasi and U. hamiltoni.

[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.

Historic importance of some aspects of research by O. Wilford Olsen on hookworms (Uncinaria lucasi) in northern fur seals (Callorhinus ursinus) and Steller sea lions (Eumatopias jubatus) in 1951 on St. Paul Island, Alaska.

The purpose of this paper is to disseminate, more widely, certain historically important aspects of research by O. Wilford Olsen on hookworms (Uncinaria lucasi) in northern fur seals (Callorhinus ursinus) and Steller sea lions (Eumatopias jubatus) in 1951 on St. Paul Island, Alaska. Highly meaningful facets of the research were recorded only in a report with limited availability by Olsen in 1952. Portions of his research presented here include measurements of hookworm free-living infective third-stage larvae (L3) and adults, photographs of bursae, and the conclusion that the species of hookworms (U. lucasi) is probably the same in both pinniped hosts. This information is especially important because very little has been published on taxonomy of hookworms in E. jubatus.

Photomicrographic images of some features of Uncinaria spp (Nematoda: Ancylostomatidae) from otariid pinnipeds.

Photomicrographs of several morphologic features of hookworms (Uncinaria spp) from northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups are presented. The main purpose is to show and describe some physical characteristics of hookworms from the two hosts; it is not to decide from these attributes whether the Uncinaria spp are the same species. The number of species of Uncinaria in pinnipeds is uncertain and specimens need to be examined from the various infected seals and sea lions before the taxonomy of these parasites can be clarified. Information in the present paper should aid in this determination.

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.