Complete mitochondrial genomes of Gnathostoma nipponicum and Gnathostoma sp., and their comparison with other Gnathostoma species.

Gnathostomiasis is a foodborne zoonotic parasitosis caused by Gnathostoma nematodes. It has caused significant public problems worldwide, but its molecular biology is limited. The purpose of this study was to decode the complete mitochondrial (mt) genomes of Gnathostoma nipponicum and Gnathostoma sp., and compare their mt sequences with other Gnathostoma species. The complete mt genome sequences were amplified by long-range PCR and determined by subsequent primer walking. The complete mt genomes of G. nipponicum and Gnathostoma sp. were 14,093bp and 14,391bp, respectively. Both of the two mt genomes contain 12 protein-coding genes (PCGs), 2 ribosomal RNA genes and 22 transfer RNA genes. The gene order and transcription direction are the same as G. spinigerum and G. doloresi. The sequence difference across the entire mt genomes varied from 14.4% to 18.2% between G. nipponicum, Gnathostoma sp., G. spinigerum and G. doloresi of Japan and China isolates. Phylogenetic analyses by Bayesian inference (BI) using concatenated amino acid sequences of 12 PCGs showed that G. nipponicum and Gnathostoma sp. are two distinctive species of Gnathostoma, and G. nipponicum are more closely related to Gnathostoma sp. than to G. spinigerum. The mtDNA datasets provide abundant resources of novel markers, which can be used for the studies of molecular epidemiology and diagnosis of Gnathostoma spp.

Effect of oral administration of Kudoa septempunctata genotype ST3 in adult BALB/c mice.

Kudoa septempunctata (Myxozoa: Multivalvulida) infects the muscles of olive flounder (Paralichthys olivaceus, Paralichthyidae) in the form of spores. To investigate the effect of K. septempunctata spores in mammals, adult BALB/c mice were fed with spores of K. septempunctata genotype ST3 (1.35 × 10(5) to 1.35 × 10(8) spores/mouse). After ingestion of spores, the mice remained clinically normal during the 24-h observation period. No spores were found in any tissue examined by histopathological screening. Quantitative PCR screening of the K. septempunctata 18S rDNA gene revealed that the K. septempunctata spores were detected only in the stool samples from the spore-fed groups. Collectively, these findings suggest that K. septempunctata spores are excreted in faeces and do not affect the gastrointestinal tract of adult mice.

The Jeju weasel, Mustela sibilica quelpartis, a new definitive host for Gnathostoma nipponicum Yamaguti, 1941.

Adult gnathostomes were discovered in the stomach of the Jeju weasel, Mustela sibilica quelpartis, road-killed in Jeju-do (Province). Their morphological characters were examined to identify the species. Total 50 gnathostome adults were collected from 6 out of 10 weasels examined. In infected weasels, 4-6 worms were grouped and embedded in each granulomatous gastric tumor, except 1 weasel. Male worms were 25.0×1.4 mm in average size, and had a tail with pedunculate papillae, a spicule, and minute tegumental spines. Females were 40.0×2.5 mm in average size, and had a tail without tegumental spines. Pointed and posteriorly curved hooklets were arranged in 8-10 rows on the head bulb. Tegumental spines were distributed from behind the head bulb to the middle portion of the body. The spines were different in size and shape by the distribution level of the body surface. Fertilized eggs were 65.5×38.9 µm in average size, and had a mucoid plug at 1 pole. These gnathostomes from Jeju weasels were identified as Gnathostoma nipponicum Yamaguti, 1941. By the present study, it was confirmed for the first time that G. nipponicum is distributed in Jeju-do, the Republic of Korea, and the Jeju weasel, M. sibilica quelpartis, plays a crucial role for its definitive host.

Discovery of larval Gnathostoma nipponicum in frogs and snakes from Jeju-do (Province), Republic of Korea.

A survey was performed to find out the intermediate hosts of Gnathostoma nipponicum in Jeju-do (Province), the Republic of Korea. In August 2009 and 2010, a total of 82 tadpoles, 23 black-spotted pond frogs (Rana nigromaculata), 7 tiger keelback snakes (Rhabdophis tigrinus tigrinus), 6 red-tongue viper snakes (Agkistrodon ussuriensis), and 2 cat snakes (Elaphe dione) were collected in Jeju-do and examined by the pepsin-HCl digestion method. Total 5 gnathostome larvae were detected in 3 (50%) of 6 A. ussuriensis, 70 larvae in 3 of 7 (42.9%) R. tigrinus tigrinus, and 2 larvae in 2 of 82 (8.7%) frogs. No gnathostome larvae were detected in tadpoles and cat snakes. The larvae detected were a single species, and 2.17 × 0.22 mm in average size. They had characteristic head bulbs, muscular esophagus, and 4 cervical sacs. Three rows of hooklets were arranged in the head bulbs, and the number of hooklets in each row was 29, 33, and 36 posteriorly. All these characters were consistent with the advanced third-stage larvae of G. nipponicum. It has been first confirmed in Jeju-do that R. nigromaculata, A. ussuriensis, and R. tigrinus tigrinus play a role for intermediate and/or paratenic hosts for G. nipponicum.

Tegumental ultrastructures of Echinoparyphium recurvatum according to developmental stages.

The present study was performed to observe tegumental ultrastructure of Echinoparyphium recurvatum according to developmental stages. Worms (1, 3, 5 and 15-day old) were recovered from chicks experimentally infected with metacercariae from Radix auricularia coreana. One-day old worms were elongated and ventrally concave, and covered with peg-like tegumental spines except the adjacent areas of the head crown and excretory pore. Type I sensory papillae were distributed on the lip of the oral sucker, and grouped ciliated papillae were around the oral sucker. Peg-like tegumental spines were densely distributed on the anterior surface of the ventral sucker level. The ventral sucker had an aspinous tegument and no sensory papillae. Tegumental spines on the posterior surface of the ventral sucker level were sparsely distributed and disappeared posteriorly. In 3 and 5-day old worms, the tegument around the oral sucker was aspinose and wrinkled concentrically. The ventral sucker had a wrinkled tegument and many bulbous papillae. Type I sensory papillae were distributed between the bulbous papillae. Tegumental spines were spade-shaped with a terminal tip. A total of 45 collar spines including 4 end group ones on both ventral corners was alternately arranged in 2 rows. The 15-day old worms were very stout and their tegumental spines were tongue-shaped without a terminal tip. From the above results, it is confirmed that the surface ultrastructure of E. recurvatum was generally similar to that of other echinostomatid flukes. However, some features, i.e., morphological change of tegumental spines and appearance of sensory papillae on the ventral sucker according to development, and number, shape and arrangement of collar spines, were characteristic, which may be of taxonomic and bioecological significance.