Intestinal helminth infections in feral cats and a raccoon dog on Aphaedo Island, Shinan-gun, with a special note on Gymnophalloides seoi infection in cats.

Four feral cats and a raccoon dog purchased from a local collector on Aphaedo Island, Shinan-gun, where human Gymnophalloides seoi infections are known to be prevalent, were examined for their intestinal helminth parasites. From 2 of 4 cats, a total of 310 adult G. seoi specimens were recovered. Other helminths detected in cats included Heterophyes nocens (1,527 specimens), Pygidiopsis summa (131), Stictodora fuscata (4), Acanthotrema felis (2), Spirometra erinacei (15), toxocarids (4), and a hookworm (1). A raccoon dog was found to be infected with a species of echinostome (55), hookworms (7), toxocarids (3), P. summa (3), and S. erinacei (1). No G. seoi was found in the raccoon dog. The results indicate that feral cats and raccoon dogs on Aphaedo are natural definitive hosts for intestinal trematodes and cestodes, including G. seoi, H. nocens, and S. erinacei. It has been first confirmed that cats, a mammalian species other than humans, play the role of a natural definitive host for G. seoi on Aphaedo Island.

Echinostome infections in the striped-field mouse, Apodemus agrarius, and the Ussuri white-toothed shrew, Crocidura lasiura, caught near the demilitarized zone, Gyeonggi-do (Province), Republic of Korea.

A total of 1,498 small mammals (rodents and insectivores), including Apodemus agrarius (n = 1,366), Crocidura lasiura (54), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutes (6), and Cricetulus triton (3), were live-trapped in Gyeonggi-do (Province) (Paju-si, Pocheon-gun, and Yeoncheon-gun) near the demilitarized zone (DMZ) from December 2004 to September 2005. A. agrarius was found to be infected with 3 species of echinostomes (Echinostoma hortense, Echinostoma cinetorchis, and Euparyphium murinum), while C. lasiura was infected with 1 species (Echinochasmus japonicas) of echinostome. Other mammals were free from echinostome infections. Total 16 E. hortense were detected in 7 (0.5%) mice, 9 E. cinetorchis from 5 (0.4%), and 3 E. murinum from 2 (0.1%) out of 1.366 A. agrarius examined. E. japonicus was found only in 1 (1.9%; total 3 specimens) C. lasiura. These results demonstrate that A. agrarius and C. lasiura, inhabiting near the DMZ of Gyeonggi-do serve as the natural definitive hosts for several species of echinostomes, although their infection rates are low. This is the first record of natural infections of A. agrarius with E. cinetorchis and C. lasiura with E. japonicus in the Republic of Korea.

Trends in parasitic diseases in the Republic of Korea.

In the Republic of Korea, cases of zoonotic, opportunistic and imported parasitoses are being detected increasingly. Vivax malaria disappeared in the late 1970s but re-emerged in 1993 and, currently, 1000-2000 cases occur annually. Brugian filariasis was endemic on offshore islands until 1990 but has now been eradicated. Soil-transmitted helminthiases (ascariasis, trichuriasis and hookworm infections) were highly prevalent until the 1970s but are now well controlled. However, food-borne trematode infections, such as clonorchiasis and intestinal trematodiases (including heterophyidiasis, echinostomiasis and gymnophalloidiasis), each show steady prevalence. This review focuses on trends in parasitic diseases in the Republic of Korea.

Gymnophalloides seoi eggs from the stool of a 17th century female mummy found in Hadong, Republic of Korea.

It was previously reported that paleoparasitological clues for parasites infecting humans could be found in the feces of mummies of the Joseon Dynasty (1392-1910) in the Republic of Korea. Here, we report the presence of trematode eggs, including Clonorchis sinensis, Metagonimus yokogawai, and Gymnophalloides seoi (a human parasite known in Korea since 1993) in the feces of a recently excavated female mummy in Hadong, Republic of Korea. This is the first report of the discovery of a G. seoi infection in a human mummy. Since Hadong is currently not an endemic area for G. seoi, we speculate that the parasite might have occurred frequently along coastal areas of the Korean peninsula several hundred years ago and that the endemic areas contracted to, more or less, restricted regions since that time.

A new gymnophallid trematode from the intestine of mice infected with metacercariae from the razor clam Sinonovacula constricta.

Meiogymnophallus sinonovaculae n. sp. (Digenea: Gymnophallidae) is described from metacercariae found in the razor clam, Sinonovacula constricta, in the Republic of Korea, and adults recovered from the small intestine of experimentally infected mice. The worms are characterized by paired clusters of vitelline follicles, a well-developed pars prostatica with abundant prostate cells, a voluminous undivided seminal vesicle (in adults and metacercariae), and large, grouped, domelike sensory papillae on the ventral surface anterior to the ventral sucker (in metacercariae). This new species resembles the type species Meiogymnophallus affinis, but differs in having compact, elliptical, and 4-5-lobed vitellaria, and an excretory vesicle with bicornuated anterior arms reaching to the oral sucker. This is the second documentation of the presence of a species of Meiogymnophallus in the Republic of Korea.

Paleoparasitological report on the stool from a Medieval child mummy in Yangju, Korea.

Previous studies have successfully shown evidence for parasitic infections in human remains from various archaeological sites. However, in the case of Korea, since there have been very few paleoparasitological reports published, pre-20th century parasitic infection patterns remain obscure. Therefore, in order to partly fill this gap, we are reporting on a case of paleoparasitic infection from the feces of a 15th century child mummy from Yangju, Korea. In the course of the present study, we found the eggs of Clonorchis sinensis, Ascaris lumbricoides, and Trichuris trichiura in the feces of the mummy. Trichuris trichiura eggs were found in far greater numbers than other parasite eggs; in fact, intact bipolar plugs were clearly observed and even the larvae were still visible in some eggs. The eggs of C. sinensis and A. lumbricoides were also well preserved, though not in as great a number. Since we could find a number of well-preserved larvae-containing eggs, we are encouraged that successful extraction, amplification, and sequence determination of ancient DNA from the paleoparasite eggs might be possible in future studies. With additional paleoparasitological investigation using feces from Korean mummies, we hope that a history of parasite infection in Korea will be reconstructed.

Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea.

Susceptibility of inbred mouse strains to Metagonimus yokogawai, Metagonimus miyatai, and Metagonimus takahashii infections was studied using BALB/c, ddY, C57BL/6J, C3H/HeN, and A/J mice, with H-2 haplotypes d, s, b, k, and a, respectively. Two hundred metacercariae were orally fed to each mouse, and the worm recovery rates (WRR), worm dimensions, and intrauterine egg numbers were measured at days 3, 7, 14, 21, and 28 postinfection (PI). On day 14 PI, the WRR of M. yokogawai was highest in ddY mice (average, 62.2%); those of M. miyatai and M. takahashii were highest in ddY (19.5%) and BALB/c mice (10.4%), respectively; worm maturation was best in C3H/HeN (M. yokogawai), C57BL/6J (M. miyatai), and ddY mice (M. takahashii). All mouse strains showed higher susceptibility to infection with M. yokogawai than with M. miyatai or M. takahashii. The results show that susceptibility of mice to Metagonimus infection varies according to mouse strain and parasite species but is suggested to be independent of the mouse H-2 haplotype.

Allelic dimorphism in the merozoite surface protein-3alpha in Korean isolates of Plasmodium vivax.

To study the genetic diversity of re-emerging Plasmodium vivax in the Republic of Korea, nucleotide sequence variations at the merozoite surface protein-3alpha (PvMSP-3alpha) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3alpha locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area.

Prevalence of Heterophyes nocens and Pygydiopsis summa infections among residents of the western and southern coastal islands of the Republic of Korea.

To determine the distribution and prevalence of heterophyid fluke infections on coastal islands of the Republic of Korea, fecal specimens were collected from 4,179 people residing on 45 islands in the West (Yellow) and South Seas and examined using the formalin-ether and Kato-Katz techniques. Eggs of Heterophyes nocens were found in 459 (11.0%) residents of 42 islands, with an average number of eggs per gram (epg) of feces of 79.6. Eggs of Pygidiopsis summa were found in 49 (1.2%) on 12 islands, with an average epg of 253.0. The egg-positive rate for H. nocens was the highest on Chungdo (32.6%), followed by Imchado (27.3%); P. summa was most prevalent on Imchado (15.2%). The majority (78.9% [362 of 549] of those infected with H. nocens and 81.6% [40 of 49] of those infected with P. summa) of those infected were adults more than 40 years old. Adult flukes of these species were recovered from residents of Imchado by treatment with praziquantel and purgation. Our results indicate that H. nocens and P. summa are indigenous to the southern and western coastal islands of the Republic of Korea.

Role of murine intestinal intraepithelial lymphocytes and lamina propria lymphocytes against primary and challenge infections with Cryptosporidium parvum.

To investigate the role of intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) in controlling Cryptosporidium parvum infection, changes in their phenotypes and functional properties were studied after induction of primary and challenge infections in immunocompetent mice. As shown by oocyst-shedding patterns, the challenge-infected group recovered more rapidly from infection than did the primary-infected group. In LPL, proportions of activated CD4+, CD25+, IgG1+, IgA+, and CD4+/IFN-gamma+ cells increased significantly in the primary-infected group compared with controls. In the challenge-infected group, proportions of these cells decreased. The antigen-specific IgA level was elevated significantly among LPL of both primary- and challenge-infected groups. Among IEL, proportions of activated CD8+, T cell receptor (TCR) gammadelta+, and CD8+/TCR gammadelta+ cells increased significantly in the challenge-infected group compared with controls and the primary-infected group; their cytotoxicity also was enhanced. However, the proportion of IEL expressing Th1 cytokines was lower than that among LPL in both infected groups. The results suggest that LPL play a more important role in protection against a primary infection with C. parvum, through the production of IFN-gamma and IgA, whereas IEL are more involved in protection against a challenge infection, through enhanced cytotoxicity.

Toll-like receptor 2 and Muc2 expression on human intestinal epithelial cells by Gymnophalloides seoi adult antigen.

Goblet cell hyperplasia and mucin hypersecretion are important for the expulsion of the intestinal trematode, Gymnophalloides seoi , from mice. However, regulatory mechanisms underlying these processes remain elusive. To better understand the effects of G. seoi antigen on the host's intestinal epithelial cells, we determined whether G. seoi induces expression of Toll-like receptors (TLRs) and mucin-related (MUC) genes on a human intestinal epithelial cell line (HT29 cells). We treated HT29 cells with G. seoi or other adult helminth antigens and measured mRNAs of TLRs and MUCs. We also performed reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry to determine whether TLR and MUC expression is regulated by interferon (IFN)-gamma, interleukin-4, or monoclonal antibodies (mAbs) against G. seoi 46 kDa antigen. Gymnophalloides seoi antigen significantly induced expression of TLR2 and MUC2 in HT29 cells, and IFN-gamma was found to upregulate TLR2 expression on the surface of the cells. The expression of MUC2 was increased by IFN-gamma, but was decreased significantly via the combination of mAbs-to-human TLRs and G. seoi antigen. These results demonstrated that G. seoi antigen upregulates TLR2 and MUC2 expression on human intestinal epithelial cells. These effects reflect a helminth-induced, IFN-gamma-dependent, and innate mucosal immune mechanism in this human intestinal cell line.

CD4+ T-cell-dependent goblet cell proliferation and expulsion of Gymnophalloides seoi from the intestine of C57BL/6 mice.

Mechanisms for the spontaneous worm expulsion from the host intestine are not well understood in gastrointestinal trematode models. We studied the role of CD4+ T-helper cells in mediating goblet cell hyperplasia and expulsion of Gymnophalloides seoi from the intestines of C57BL/6 (resistant) and ICR (susceptible) mice. C57BL/6 mice expelled all G. seoi worms within 4 days post-infection (PI), while ICR mice did not completely expel worms until day 7 PI. This difference in worm expulsion was associated with high numbers of mucosal goblet cells in C57BL/6 mice along with alteration of the mucin quality, with changes in the terminal sugar chain and high levels of IL-4 and IL-5 mRNA expression in mesenteric lymph nodes. Adoptive transfer of mucosal CD4+ T-helper cells to syngeneic mice elicited strong goblet cell hyperplasia and a notably accelerated worm expulsion. However, this T-helper cell transfer had no relationship with the alteration of mucin quality. The results showed that CD4+ T-helper cells play an important role as a mediator of goblet cell hyperplasia, but not for functional activation of goblet cells. It is suggested that both T-cell dependent and independent mechanisms operate for expulsion of G. seoi from the mouse intestine.

The ruddy turnstone, Arenaria interpres interpres, a new definitive host for Gynaecotyla squatarolae (Digenea: Microphallidae).

The ruddy turnstone, Arenaria interpres interpres, a migratory Korean bird, was proved to be a natural definitive host for Gynaecotyla squatarolae (Digenea: Microphallidae). The ruddy turnstone was found dead at the seashore of Okgueup, Gunsan-si, Jeollabuk-do. The intestinal tract was examined, and 98 unknown flukes were recovered. The worms were 600 284 micrometer in size, and had 2 ventral suckers. The seminal vesicle was large, the genital atrium was prominent, and the average egg size was 20 12.5 micrometer. Based on these results, the worms were identified as G. squatarolae. This is the first report on the ruddy turnstone as a natural definitive host of G. squatarolae in the Republic of Korea.

Antibody responses to Cryptosporidium antigen in HIV-positive patients in the Republic of Korea.

The diagnosis of cryptosporidiosis has been carried out using coprologic techniques in the Republic of Korea. However, antibody responses to Cryptosporidium have rarely been studied. Serum antibodies from HIV-positive/oocyst-positive Korean patients recognized significantly 31 and 27 kDa antigens, and HIV-negative/oocyst-positive individuals clearly reacted to 15/17 kDa antigens. Compared with oocyst-positive cases, 18.7% and 75.8% of sera from HIV-positive patients reacted to 31 and 27 kDa antigens. Only 11.1% of HIV-negative individuals reacted to 15/17 kDa. Based on these findings, serum antibody responses were different between HIV-positive and HIV-negative individuals infected with Cryptosporidium, and it is suggested that HIV-positive patients are more frequently exposed to C. parvum compared to HIV-negative individuals.

Holostephanus metorchis (Digenea: Cyathocotylidae) from chicks experimentally infected with metacercariae from a fish, Pseudorasbora parva, in the Republic of Korea.

Holostephanus metorchis (Digenea: Cyathocotylidae) is a parasite of birds, transmitted by freshwater fishes. H. metorchis adults were recovered from chicks experimentally infected with metacercariae collected from freshwater fishes, Pseudorasbora parva. The metacercariae were oval, surrounded with thick fibrous capsules. In adult flukes, the holdfast organ occupied the ventral concavity, and the anterior testis did not reach the level of the ventral sucker. Based on these morphological characteristics, these flukes were identified as H. metorchis.

Microphallus koreana n. sp. (Trematoda: Microphallidae) transmitted by a marine crab, Macrophthalmus dilatatus.

Microphallus species occur primarily as intestinal parasites of birds and mammals, and metacercariae of a new species belonging to this genus have been discovered from the crab, Macrophthalmus dilatatus, in the Republic of Korea. The metacercaria of this fluke was round with 2 thick walls, and the excysted one had mature genital organs. The adult flukes recovered from experimentally infected chicks had numerous intrauterine eggs, well-developed pars prostatica, widely bifurcating ceca, and prominent uterine bulge. After observing internal structures, it was concluded that this species is different from any other known Microphallus spp. Based on the morphology of metacercariae and adult flukes, we describe this specimen as a new species, Microphallus koreana n. sp.

Tegumental ultrastructure of adult Gynaecotyla squatarolae (Digenea: Microphallidae).

Gynaecotyla squatarolae (Digenea: Microphallidae) adult flukes were recovered from experimental chicks at day 4-6 post-infection and their tegumental ultrastructure was observed with a scanning electron microscopy. They were pyriform in shape, and their anterior halves were concaved ventrally. The whole body surface was covered with tegumental spines, which were wide and 16-17 digitated between oral and ventral suckers. The density of spines and number of digits decreased posteriorly. The oral sucker was subterminal and the excretory pore was at the posterior end of the worm. Two ventral suckers were similar in appearance and protruded near midline of the worm. The genital atrium was dextral to the small ventral sucker. The dorsal surface was covered with tegumental spines, but the spines were sparser than on the ventral surface. On the middle portion of the dorsal surface, a small opening presumed to be the Laurer's canal was seen. From these findings, it has been confirmed that the adult G. squatarolae has unique characteristics in the surface ultrastructure.

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum.

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.

Gynaecotyla squatarolae (Digenea: Microphallidae) from rats experimentally infected with metacercariae from the shore crab, Macrophthalmus dilatatus.

Metacercariae of Gynaecotyla squatarolae (Digenea: Microphallidae) were discovered from the shore crab, Macrophthalmus dilatatus, purchased at a market in a coastal town of Taean-Eup, Chungcheongnam-do, Republic of Korea. Their adult flukes were confirmed by experimental infection of rats. The metacercariae of G. squatarolae were elliptical (391.1 x 362.5 micrometer), and the excysted metacercariae had progenetic genital organs, including the ovary and testes. To obtain adult flukes, 6 Sprague-Dawley rats were fed 500 metacercariae each, and killed at days 2, 4, and 6 post-infection. The adult flukes were identified as G. squatarolae (Yamaguti, 1934) Yamaguti, 1939, based on morphological characters, including 2 ventral suckers (1 large and 1 small), a large genital atrium equipped with the cirrus and the metraterm, separated male and female genital pores, a transversely long cirrus pouch, and extensive vitelline follicles. In the present study, it has been first proven that the shore crab M. dilatatus is a second intermediate host for G. squatarolae in the Republic of Korea.

A new endemic focus of Heterophyes nocens and other heterophyid infections in a coastal area of Gangjin-gun, Jeollanam-do.

To know the prevalence of heterophyid trematodes among inhabitants of a southern coastal village, i.e., Sacho-ri, Gangjin-gun, Jeollanam-do (Province), 82 stool samples were examined on helminth eggs and protozoan cysts using Kato-Katz and formalin-ether sedimentation techniques. Total 33 people (40.2%) were positive for trematodes (Heterophyes nocens 15 people, Pygidiopsis summa 3, Metagonimus sp.; 4, Clonorchis sinensis 7, Gymnophalloides seoi 6) and/or protozoa (Entamoeba coli 3). Among intestinal trematode egg positive cases, 17 were treated with praziquantel and their whole diarrheic stools were collected after purgation. Adult flukes of H. nocens (number of specimens = 1,294), P. summa (386), Stellantchasmus falcatus (5), Stictodora lari (4), and Heterophyopsis continua (1) were collected using a stereomicroscope. To know the source of human H. nocens infections in this village, metacercarial infections in mullets (10) were examined and most H. nocens metacercariae (101/105, 96.2%) were found in the trunk portion. From above results, the surveyed coastal village has been newly known as an endemic focus of human H. nocens infection and consuming raw mullets was the presumable source of human heterophyid infections.