The phylogenetic relationships of the 3 Neodiplostomum spp. (Digenea: Neodiplostomidae) occurring in Korea (N. seoulense, N. leei, and N. boryongense) were analyzed using the partial mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. The adult flukes were recovered from Sprague-Dawley rats (N. seoulense) and newborn chicks (N. leei and N. boryongense) experimentally infected with the neodiplostomula from the grass snake, Rhabdophis tigrinus tigrinus. The genomic DNA was amplified using specific primers, and the sequence of CO1 was obtained. According to the results, the pairwise similarity was 96.1% between N. boryongense and N. seoulense, but was 95.0% between N. boryongense and N. leei and 94.2% between N. leei and N. seoulense. The results demonstrated a closer phylogenetic relationship between N. seoulense and N. boryongense. This high relationship of N. seoulense and N. boryongense may be related to their similar morphologic features including the limited distribution of vitellaria and the presence of a genital cone. N. leei is distinct on the other hand with an extensive distribution of vitellaria and the absence of a genital cone.

Neodiplostomum seoulense (Digenea: Neodiplostomidae) is an intestinal trematode that can cause severe mucosal pathology in the small intestines of mice and even mortality of the infected mice within 28 days after infection. We observed neuronal growth associated protein-43 (GAP-43) expression in the myenteric plexus of the small intestinal wall of N. seoulense-infected mice until day 35 post-infection (PI). BALB/c mice were infected with 200 or 500 N. seoulense metacercariae isolated from naturally infected snakes and were killed every 7 days for immunohistochemical demonstration of GAP-43 in the small intestines. N. seoulense-infected mice showed remarkable dilatation of intestinal loops compared with control mice through days 7-28 PI. Conversely, GAP-43 expression in the mucosal myenteric plexus was markedly (P<0.05) reduced in the small intestines of N. seoulense-infected mice during days 7-28 PI and was slightly normalized at day 35 PI. From this study, it is evident that neuronal damage occurs in the intestinal mucosa of N. seoulense-infected mice. However, the correlation between intestinal pathology, including the loop dilatation, and depressed GAP-43 expression remains to be elucidated.

To examine humoral immune responses in the host, we measured serum antibody levels in different strains of mice (ICR, BALB/c, and C3H) experimentally infected with Neodiplostomum seoulense. Specific IgG antibody levels were increased remarkably with little difference among 3 strains of mice infected with N. seoulense from day 7 to 35 post-infection. More target proteins of adult parasites reacted with IgG at the time when the worm recovery decreased compared with other times. More than 20 protein bands, from 14 kDa to 94 kDa in size, were separated from the crude antigen of N. seoulense adults by SDS-PAGE, and among them 26, 30, 35, 43, 54, 67, and 94 kDa proteins were the major antigenic proteins. The results suggest that significant IgG antibody responses occur against N. seoulense in mice and this may be related with expulsion of worms.

A total of 1,496 rodents and insectivores were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804), and Pocheon-gun (343), Gyeonggi-do (Province), and examined for intestinal helminths, including Neodiplostomum seoulense, seasonally from December 2004 to September 2005. Six species of rodents, including Apodemus agrarius (1,366), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutus (6), and Cricetulus triton (3), and 1 species of insectivores Crocidura lasiura (54) were collected. A total of 321 adult N. seoulense were collected from 19 (1.4%) A. agrarius. The worm burden ranged from 1 to 101 per A. agrarius (mean; 16.9). No N. seoulense was observed in other rodent or insectivore species examined. The infection rate during autumn (4.5%) was higher than those during spring (0.8%), summer (0.8%), and winter (0.5%). The average number of N. seoulense in infected A. agrarius was the highest in spring (66.0 specimens), followed by autumn (15.2), winter (4.5), and summer (3.3). This study first confirms that A. agrarius is a natural definitive host for N. seoulense, and demonstrates that the infection rates and intensities vary seasonally and geographically.

The furcocercus cercariae of Neodiplostomum seoulense (Digenea: Neodiplostomidae) penetrate the skins of tadpoles and shed their tails. The speculated mechanism of this tail loss was physical efforts required to produce a vigorous zigzag motion during skin penetration; no other mechanism has been proposed. We examined the relationship between the host serum and cercarial tail loss. Cercariae of N. seoulense were collected from experimentally infected Segmentina hemisphaerula, and lots of 300 cercariae were cultured in medium 199 contained several types of sera. Cercarial tail degradation was induced in all media, but all the cercariae cultured except those cultured in media containing fetal bovine serum (FBS) died within 48 hr. After 72 hr cultivation in media containing FBS, cercarial tail degradation was induced in 67.0%; in continuous cultivation 13.3% of larvae survived for 7 days. Tail degradation did not occur in the absence of serum and when serum was heat inactivated at 56℃ for 30 min. The addition of 20 mM ethylenediaminetetraacetic acid (EDTA) blocked cercarial tail degradation completely. Moreover, the addition of 20 mM MgCl₂ restored tail degradation blocked by EDTA. These results suggest that the alternative complement pathway is related with the N. seoulense cercarial tail degradation induced by serum.

The effects of anti-allergic drugs on intestinal mastocytosis and the expulsion of Neodiplostomum seoulense were observed in Sprague-Dawley rats, after oral infection with 500 metacercariae. The drugs used were hydroxyzine (a histamine receptor H₁ blocker), cimetidine (a H₂ blocker), cyclosporin-A (a helper T-cell suppressant), and prednisolone (a T- and B-cell suppressant). Infected, but untreated controls, and uninfected controls, were prepared. Worm recovery rate and intestinal mastocytosis were measured on weeks 1, 2, 3, 5, and 7 post-infection. Compared with the infected controls, worm expulsion was significantly (P < 0.05) delayed in hydroxyzine- and cimetidine-treated rats, despite mastocytosis being equally marked in the duodenum of all three groups. In the cyclosporin-A- and prednisolone-treated groups, mastocytosis was suppressed, but worm expulsion was only slightly delayed, without statistical significance. Our results suggest that binding of histamine to its receptors on intestinal smooth muscles is more important in terms of the expulsion of N. seoulense from rats than the levels of histamine alone, or mastocytosis.

A karyotype analysis of the chromosome of Neodiplostomum seoulense, one of causative agents of human intestinal trematodiasis, was done from the gonad tissue by the squashing method. The chromosome number was n = 10 and 2n = 20. Chromosome length was 1.30-4.0 ?m. Chromosome pairs in the complement consisted of two pairs of metacentric, five pairs of submetacentrics/subtelocentric and three pairs of telocentric chromosomes. These data were comparable with those of other intestinal trematodes.