Fecundity reduction of BALB/c mice after survival from lethal Neodiplostomum seoulense infection.

Neodiplostomum seoulense (Digenea: Neodiplostomidae), an intestinal trematode infecting humans and rodents, is known to be highly pathogenic and lethal to experimentally infected mice. Only a small proportion of mice can survive from its infection. This study aimed to assess the reproductive capacity of surviving BALB/c mice. The fertility of male and female mice, birth time (period from mating to birth of litters), number of litters, size and weight of testes or ovary-oviduct-uterus, apoptosis of testicular cells, and serum levels of sex hormones were determined. Our results revealed that surviving mice underwent severe fecundity reduction and finally became infertile. They could not be able to produce generations beyond F4. Fertility rate, birth time, and number of litters of N. seoulense-infected mice were all significantly (p < 0.05) lower than those of uninfected controls, Metagonimus miyatai (less pathogenic intestinal trematode)-infected, or castor oil (severe diarrheal agent)-administered controls. The size and weight of testes or ovary-oviduct-uterus were markedly (p < 0.05) decreased after N. seoulense infection. Moreover, the number of apoptotic cells in the testicular tissue was significantly (p < 0.05) increased (up to 10-50-folds) during weeks 1-3 post-infection. Serum testosterone levels in infertile mice were reduced to 1/10 level of fertile mice. These results indicated that BALB/c mice surviving N. seoulense infection underwent destruction and apoptosis of gonad tissues with fecundity reduction. They were finally infertile, with no ability to produce their next generations.

Seroprevalence of Trichinella sp. in Wild Boars (Sus scrofa) from Yanggu-gun, Gangwon-do, Korea.

A total 7 outbreaks of trichinellosis have occurred in Korea, mostly as a result of consumption of raw wild boar (Sus scrofa) meat. Since only 1 serological survey on wild boars had yet been performed in Korea, the present study aimed to estimate the prevalence of trichinellosis in wild boars and some species of rodents by artificial digestion and serological examinations in Yanggu-gun, Gangwon-do, the endemic area of trichinellosis. Both the wild boar and rodent muscle samples revealed no Trichinella larvae by direct examination and artificial digestion method. However, serological examinations revealed that 4 wild boar sera samples out of 118 (3.4%) were positive to Trichinella antigen. Although the recovery of Trichinella larvae ended in a failure, it is proved for the first time that the sylvatic cycle of Trichinella has been maintained in wild boars of Gangwon-do, Korea.

Intestinal nematodes from small mammals captured near the demilitarized zone, Gyeonggi province, Republic of Korea.

A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.

Four human cases of Acanthotrema felis (Digenea: Heterophyidae) infection in Korea.

Acanthotrema felis is an intestinal trematode of cats originally reported from the Republic of Korea. Only 1 human case infected with a single adult worm has been previously recorded. In the present study, we report 4 human cases infected with a total of 10 worms recovered after anthelmintic treatment and purging. All 4 patients reside in coastal areas of Jeollanam-do, Korea, and have consumed brackish water fish including the gobies, Acanthogobius flavimanus. The worms averaged 0.47 mm in length and 0.27 mm in width, and had 3 sclerites on the ventrogenital sac; 1 was short and thumb-like, another was long and blunt-ended, and the 3rd was long and broad-tipped. They were identified as A. felis Sohn, Han, & Chai, 2003. Surveys on coastal areas to detect further human cases infected with A. felis are required.

Immune responses of mice intraduodenally infected with Toxoplasma gondii KI-1 tachyzoites.

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1 × 10(5) tachyzoites) or day 11 PI (with 1 × 10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4(+) and CD8(+) T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-γ and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-α was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-γ, TNF-α, and IL-10, in response to T. gondii infection.

Suppression of CD4 T-Cells in the spleen of mice infected with Toxoplasma gondii KI-1 tachyzoites.

Toxoplasma gondii KI-1, a recent new isolate from Korea, shows similar pathogenicity and infectivity to mice compared to the virulent RH strain. To understand characteristics of host immunity, including immune enhancement or suppression, we investigated proliferative responses and phenotypes of spleen cells. In addition, kinetics of IFN-γ, a Th1 cytokine, was examined in BALB/c mice up to day 6 post-infection (PI). Intraperitoneal injection of mice with 10(3) KI-1 tachyzoites induced significant decreases (P < 0.05) in proliferative responses of spleen cells. This occurred at days 2-6 PI even when concanavalin A (con A) was added and when stimulated with KI-1 antigen, suggesting suppression of the immunity. CD4(+) T-cells decreased markedly at day 2 PI (P < 0.05), whereas CD8(+) T-cells, NK cells, and macrophages did not show significant changes, except a slight, but significant, increase of CD8(+) T-cells at day 6 PI. The capacity of splenocytes to produce IFN-γ by con A stimulation dropped significantly at days 2-6 PI. These results demonstrate that intraperitoneal injection of KI-1 tachyzoites can induce immunosuppression during the early stage of infection, as revealed by the decrease of CD4(+) T-cells and IFN-γ.

Infection status of freshwater crabs and crayfish with metacercariae of Paragonimus westermani in Korea.

The present study investigated the infection status of Paragonimus westermani metacercariae in freshwater crabs (n = 363) and crayfish (n = 31) from October 2007 to October 2008 using the crush method. All of the freshwater crabs, Eriocheir japonicus, were negative for P. westermani metacercariae while 10 (32.3%) of the 31 examined crayfish were positive. The 10 positive crayfish were caught in Haenam, Jeollanam-do, and there were 8-59 (mean 28.4) metacercariae per infected crayfish. These results suggest that P. westermani metacercariae are still transmitted by crayfish enzootically in southern Korea, and that freshwater crabs may transmit metacercariae only on rare occasions.

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.

A new neodiplostomid (Digenea) from the intestine of chicks infected with metacercariae from the grass snake, Rhabdophis tigrina.

Three species of neodiplostomula are known to inhabit the European grass snake, Rhabdophis tigrina, in the Republic of Korea: Pharyngostomum cordatum (large-sized neodiplostomula), an intestinal trematode of cats; Neodiplostomum seoulense (small-sized neodiplostomula), an intestinal trematode of humans and rodents; and Neodiplostomum leei (small-sized neodiplostomula), which migrates to the livers of rodents and is an intestinal trematode of birds. The present study describes a fourth species, Neodiplostomum (Conodiplostomum) boryongense n. sp. (Digenea: Neodiplostomidae), based on adult flukes recovered from the small intestines of chicks experimentally infected with small-sized neodiplostomula from the grass snake. The new species differs from 13 previously known species. It also differs from N. seoulense in its larger body size, severely bilobed testes, and smaller genital atrium, and from N. leei in its larger body size, smaller ventral sucker, presence of a genital cone, and vitelline follicles distributed chiefly in the forebody. The new species does not migrate to the livers of rodents nor does it develop to adulthood in the rodent intestines. However, the neodiplostomula of the new species are indistinguishable from those of the other 2 species. Results show that at least 4 species of neodiplostomula inhabit the grass snake in the Republic of Korea.

T-helper-1 and T-helper-2 immune responses in mice infected with the intestinal fluke Neodiplostomum seoulense: their possible roles in worm expulsion and host fatality.

Neodiplostomum seoulense is highly pathogenic and lethal to experimental mice; most worms are expelled within 2 mo of acquisition. In this study, T-helper (Th) cell immune responses were studied in N. seoulense-infected BALB/c mice. Spleen and mesenteric lymph node (MLN) cells of infected mice proliferated in response to parasite antigens; CD4+ T cells proliferated more than CD8+ T cells. Antigen-induced interferon (IFN)-gamma (a Th1 cytokine) secretion began to increase at day 7 postinfection (PI) in spleen and MLN cells, and this was maintained at day 28 PI, whereas interleukin (IL)-4 (a Th2 cytokine) secretion was somewhat lower. Similar results were observed for mRNA signals of IFN-gamma and IL-4. Antigen-specific serum total immunoglobulin (Ig)G, IgG1, IgM, and IgA levels (Th2-induced) were elevated from days 7 to 14 to day 28 PI, and IgG2a (Th1-induced) was elevated at days 21 to 28 PI. Interestingly, the numbers of macrophages (Th1- or Th2-induced), which were found to kill N. seoulense worms in vitro, increased remarkably during days 14-28 PI in spleens and small intestines of infected mice. This study shows that mixed Th1 and Th2 responses occur during the course of N. seoulense infection in BALB/c mice. Heavy infiltrations of macrophages in the small intestine may participate in host damage and worm expulsion.

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.

Migration of Neodiplostomum leei (Digenea: Neodiplostomidae) neodiplostomula to the livers of various mammals.

Of morphologically indistinguishable small-sized neodiplostomula in the grass snake Rhabdophis tigrina in the Republic of Korea, some, such as Neodiplostomum seoulense, mature into adults in the intestines of rodents, whereas others, such as Neodiplostomum leei, migrate to rodent livers and mature in the intestines of chicks. In the present study, we aimed to observe in more detail the extraintestinal migration and development of N. leei by using various animal models, i.e., mice, rats, hamsters, rabbits, cats, and chicks. In mammals, small-sized neodiplostomula (N. leei) inoculated orally penetrated the intestinal wall, entered the peritoneal cavity, and oriented to the liver without passing through any other organ. In rodent livers, the neodiplostomula were surrounded by host inflammatory cells and fibrotic tissues from day 10 postinfection (PI); the worms were found dead by day 56 PI. When neodiplostomula from rodent livers were transferred orally to mammals, they reoriented to the liver, although they were able to develop into adults in the chick intestine by day 6-7 PI. The possibility of human infections, i.e., liver migration, by N. leei neodiplostomula, among snake consumers, warrants investigation.

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.

Heterophyid trematodes recovered from people residing along the Boseong River, South Korea.

We conducted an epidemiological survey to determine the status of heterophyid fluke infections among people residing along the Boseong River, Gokseong-gun, South Korea (= Korea) from October 2011 to February 2012. Fecal specimens were collected from 115 (male 51, female 64) people and examined for intestinal helminth eggs using the Kato-Katz thick smear technique. The eggs of Metagonimus yokogawai together with other Metagonimus spp. were detected in 28 (24.3%) cases. Eleven egg positive people were treated with 10mg/kg praziquantel followed by MgSO4 purging in order to recover the adult flukes. Whole consecutive diarrheic stools were collected individually 4-5 times. Adult flukes recovered were 66,499 specimens (6045.4/positive case) of M. yokogawai, 343 (38.1) of Metagonimus miyatai, 3293 (299.4) of Metagonimus takahashii, 81 (20.3) of Heterophyes nocens, 6 (3.0) of Heterophyopsis continua, and 1 (1.0) of Stictodora fuscata. The results indicated that the surveyed area is a highly endemic area of metagonimiasis (three Metagonimus species) with low-grade mixed infections of 3 other heterophyid flukes. The infected people experienced variable degrees of gastrointestinal discomfort and indigestion. They consumed raw freshwater and brackish water fish, including sweetfish and mullets. It is strongly recommended that people residing in the survey area avoid eating raw fish to prevent M. yokogawai and other heterophyid infections.

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.

Centrocestus formosanus (Digenea: Heterophyidae) encysted in the freshwater fish, Puntius brevis, from Lao PDR.

The metacercariae of Centrocestus formosanus, a minute intestinal trematode of mammals and birds, were detected in the freshwater fish, Puntius brevis, from Vientiane Municipality, Lao PDR. The metacercariae were experimentally fed to mice, and adult flukes were recovered in their small intestines 7 days later. The adult flukes were morphologically characterized by having 32 (rarely 34) circumoral spines arranged in 2 alternative rows, a large bipartite seminal vesicle, an oval-shaped ovary, and an X-shaped excretory bladder. Based on these characters, the adults were identified as Centrocestus formosanus (Nishigori, 1924). The taxonomic significance of C. formosanus, in relation to a closely related species, C. caninus (Leiper, 1913), is briefly discussed. It has been first verified by adult worm recovery that C. formosanus is prevalent in Vientiane areas of Lao PDR, taking the freshwater fish, P. brevis, as a second intermediate host.

Infection status of freshwater fish with metacercariae of Clonorchis sinensis in Korea.

This study investigated freshwater fish for their current infection status with metacercariae of Clonorchis sinensis in Korea. Twenty-one species of freshwater fish (n = 677) were collected from 34 regions nationwidely from February 2007 to June 2008. They were individually examined by digestion technique. Eight species of freshwater fish from 17 different regions were recognized positive for the metacercariae of C. sinensis. The positive rates (range of metacercariae number per fish) of fish by the species were as follows: 48% (1-1,142) in Pseudorasbora parva, 60% (1-412) in Pungtungia herzi, 15.7% (1-23) in Pseudogobio esocinus, 29% (1-7) in Acheilognathus intermedia, 21% (1-4) in Odontobutis interrupta, 33% (1-6) in Zacco temmincki, 3.6% (1-4) in Zacco platypus, and 26.3% (1) in Hemibarbus labeo. The two species, P. parva and P. herzi, are able to be the index fish for estimation of C. sinensis transmission in a certain locality. Still several species of freshwater fish are briskly transmitting C. sinensis infection in many riverside areas of southern Korea.

Decrease of Metagonimus yokogawai endemicity along the Tamjin River basin.

The Tamjin River which flows from Jangheung-gun via Gangjin-gun to the South Sea was reported to be a highly endemic area of Metagonimus yokogawai infection in 1977 and 1985. However, there were no recent studies demonstrating how much change occurred in the endemicity, in terms of prevalence and worm burden, of metagonimiasis in this river basin. Thus, a small-scale epidemiological survey was carried out on some residents along the Tamjin River basin in order to determine the current status of M. yokogawai infection. A total of 48 fecal samples were collected and examined by the Kato-Katz thick smear and formalin-ether sedimentation techniques. The egg positive rate of all helminths was 50.0%, and that of M. yokogawai was 37.5%, followed by C. sinensis 22.9% and G. seoi 4.2%. To obtain the adult flukes of M. yokogawai, 6 egg positive cases were treated with praziquantel 10 mg/kg in a single dose and purged with magnesium sulfate. A total of 5,225 adult flukes (average 871 specimens per person) of M. yokogawai were collected from their diarrheic stools. Compared with the data reported in 1977 and 1985, the individual worm burdens appeared to have decreased remarkably, although the prevalence did not decrease at all. It is suggested that the endemicity of M. yokogawai infection along the Tamjin River has been reduced. To confirm this suggestion, the status of infection in snail and fish intermediate hosts should be investigated.

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.