Echinostoma ilocanum infection in Oddar Meanchey Province, Cambodia

Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.     

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

Echinostoma revolutum: metacercariae in Filopaludina snails from Nam Dinh Province, Vietnam, and adults from experimental hamsters

We detected metacercariae of Echinostoma revolutum in Filopaludina sp. snails purchased from a local market in Nam Dinh Province for the first time in Vietnam. Adult flukes were harvested from experimentally infected hamsters at days 14 and 17 post-infection. The metacercariae were round, 170-190 μm (n = 15) in diameter, with a cyst wall thickness of about 12 μm. A total of 37 collar spines were arranged around the head collar, and large excretory granules were seen in 2 canals of the excretory bladder. The 14-day old adult flukes were elongated, ventrally curved, and 5.0-7.2 × 0.8-1.3 mm (n = 20). The head collar had a total of 37 collar spines arranged in 2 alternating rows, including 5 corner spines on each side. The cirrus sac contained a saccular seminal vesicle, a prostatic gland, and an unarmed cirrus. Two tandem testes were smooth or slightly lobed. Eggs were ovoid to elliptical, 110-118 × 70-75 μm. These morphological characters were similar to those of E. revolutum and E. jurini. We tentatively identified it as E. revolutum because the validity of E. jurini remains to be elucidated. The taxonomic relationship of E. revolutum and E. jurini is discussed.     

A Case of Echinostoma cinetorchis (Trematoda: Echinostomatidae) Infection Diagnosed by Colonoscopy

Human cases of echinostomiasis have been sporadically diagnosed by extracting worms in the endoscopy in Korea and Japan. Most of these were caused by Echinostoma hortense infection. However, in the present study, we detected 2 live worms of Echinostoma cinetorchis in the ascending colon of a Korean man (68-year old) admitted to the Gyeongsang National University Hospital with complaint of intermittent right lower quadrant abdominal pain for 5 days. Under colonoscopy, 1 worm was found attached on the edematous and hyperemic mucosal surface of the proximal ascending colon and the other was detected on the mid-ascending colon. Both worms were removed from the mucosal surface with a grasping forceps, and morphologically identified as E. cinetorchis by the characteristic head crown with total 37 collar spines including 5 end-group ones on both sides, disappearance of testes, and eggs of 108×60 µm with abopercular wrinkles. The infection source of this case seems to be the raw frogs eaten 2 months ago. This is the first case of endoscopy-diagnosed E. cinetorchis infection in Korea.     

Complete Mitochondrial Genome of Echinostoma hortense (Digenea: Echinostomatidae)

Echinostoma hortense (Digenea: Echinostomatidae) is one of the intestinal flukes with medical importance in humans. However, the mitochondrial (mt) genome of this fluke has not been known yet. The present study has determined the complete mt genome sequences of E. hortense and assessed the phylogenetic relationships with other digenean species for which the complete mt genome sequences are available in GenBank using concatenated amino acid sequences inferred from 12 protein-coding genes. The mt genome of E. hortense contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region. The length of the mt genome of E. hortense was 14,994 bp, which was somewhat smaller than those of other trematode species. Phylogenetic analyses based on concatenated nucleotide sequence datasets for all 12 protein-coding genes using maximum parsimony (MP) method showed that E. hortense and Hypoderaeum conoideum gathered together, and they were closer to each other than to Fasciolidae and other echinostomatid trematodes. The availability of the complete mt genome sequences of E. hortense provides important genetic markers for diagnostics, population genetics, and evolutionary studies of digeneans.     

A human infection of Echinostoma hortense in duodenal bulb diagnosed by endoscopy

As gastroduodenoscopy performed more frequently, case reports of human echinostomiasis are increasing in Korea. A Korean woman presented at a local clinic with complaints of abdominal pain and discomfort that had persisted for 2 weeks. Under gastroduodenoscopy, two motile flukes were found attached on the duodenal bulb, and retrieved with endoscopic forceps. She had history of eating raw frog meat. The two flukes were identified as Echinostoma hortense by egg morphology, 27 collar spines with 4 end-group spines, and surface ultrastructural characters. This report may prove frogs to be a source of human echinostome infections.     

Corbicula fluminea (Bivalvia: Corbiculidae): a possible second molluscan intermediate host of Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea.

More than 1,500 clams of Corbicula fluminea, the most favorable food source of freshwater bivalves in Korea, were collected from 5 localities to examine cercarial and metacercarial infection with Echinostoma cinetorchis. Although 3 clams infected with suspicious E. cinetorchis metacercariae out of 200 specimens collected at Kangjin, Chollanam-do were detected, no cercarial and metacercarial infections with E. cinetorchis were observed in field-collected Corbicula specimens. In the susceptibility experiments with laboratory-reared clams, those infected with miracidia of E. cinetorchis did not release their cercariae up to 60 days after infection. To confirm the identity of second intermediate host of E. cinetorchis experimentally, a total of 30 clams were exposed to the cercariae from Segmentina hemisphaerula that had been infected with miracidia of E. cinetorchis. The clams were susceptible to cercariae of E. cinetorchis with an infection rate of 93.3%. Metacercariae from clams taken more than 7 days after cercarial exposure were fed to rats (S/D strain), and adult worms of E. cinetorchis, characterized by 37-38 collar spines on the head crown, were recovered from the ileocecal regions. This is the first report of C. fluminea as a possible second intermediate host of E. cinetorchis.     

Austropeplea ollula (Pulmonata: Lymnaeidae): a new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

Three freshwater snail species of the family Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula and Fossaria truncatula. Out of 3 lymnaeid snail species, A. ollula was naturally infected with the Echinostoma cinetorchis cercariae (infection rate = 0.7%). In the experiments with the laboratory-bred snails, F. truncatula as well as A. ollula was also susceptible to the E. cinetorchis miracidia with infection rates of 25% and 40%, respectively. All of three lymnaeid snail species exposed to the E. cinetorchis cercariae were infected with the E. cinetorchis metacercariae. It is evident that A. ollula acts as the first molluscan intermediate host of E. cinetorchis in Korea, and F. truncatula may be a possible candidate for the first intermediate host of this intestinal fluke. Also, three lymnaeid snail species targeted were experimentally infected with E. cinetorchis metacercariae.     

Echinostoma revolutum: Freshwater Snails as the Second Intermediate Hosts in Chiang Mai,Thailand

The occurrence of 37-collar spined echinostome metacercariae in freshwater snails was investigated in 6 districts of Chiang Mai Province, Thailand, from October 2011 to April 2012. A total of 2,914 snails that belong to 12 species were examined, and 7 snail species (Clea helena, Eyriesia eyriesi, Bithynia funiculata, Bithynia siamensis siamensis, Filopaludina doliaris, Filopaludina sumatrensis polygramma, and Filopaludina martensi martensi) were found infected with echinostome metacercariae. The prevalence of metacercariae was the highest in Filopaludina spp. (38.5-58.7%) followed by B. funiculata (44.0%), E. eyriesi (12.5%), B. siamensis siamensis (8.2%), and C. helena (5.1%). Metacercariae were experimentally fed to hamsters and domestic chicks, and adult flukes were recovered from both hosts at days 15 and 20 post-infection. The adult flukes were identified based on morphological features, morphometrics, host-parasite relationships, and geographical distribution. They were compatible to Echinostoma revolutum or Echinostoma jurini, with only minor differences. As the adults were recovered from both hamsters and chicks, our specimens were more compatible to E. revolutum rather than E. jurini (reported only from mammals). This is the first report for metacercariae of E. revolutum in the snail host, C. helena, and also confirmed that Filopaludina spp., E. eryresi, and Bithynia spp. act as the second intermediate hosts of E. revolutum under natural conditions, which are indigenously distributed in Chiang Mai province.     

Echinostoma macrorchis in Lao PDR: Metacercariae in Cipangopaludina Snails and Adults from Experimentally Infected Animals

The echinostome metacercariae encysted in Cipangopaludina sp. snails that were purchased from a market in Vientiane Municipality, Lao PDR, were identified as Echinostoma macrorchis (Digenea: Echinostomatidae) through recovery of adult flukes after experimental infection to rats and a cat. The metacercariae were round, 113-128 (121)×113-125 (120) µm, having a thick cyst wall, a head collar armed with collar spines, and excretory granules. The adult flukes recovered from the rats and cat at day 14 and 30 post-infection, respectively, were elongated, ventrally curved, and 3.9-6.3×0.7-1.1 mm in size. The head collar was distinct, bearing 43-45 collar spines with 5 angle spines on each side. Two testes were large (as the name implies), tandem, and slightly constricted at the middle, with irregular margins. Eggs were operculated, ovoid to elliptical, and 88-95×56-60 µm. In scanning electron microscopy, the head collar was prominent, with 43-45 collar spines. Scale-like tegumental spines were densely distributed on the ventral surface between the oral and ventral suckers. Sensory papillae were distributed mainly on the tegument around the 2 suckers. It is confirmed that E. macrorchis is distributed in Lao PDR using Cipangopaludina sp. snails as the second intermediate host.     

Mucosal mast cell responses in the small intestine of rats infected with Echinostoma hortense

Mucosal mast cell (MMC) responses and worm recovery rates in rats infected with Echinostoma hortense were investigated from day 3 to day 56 post-infection (p.i.). Experimental infected group showed apparently higher number of MMC in each part of the small intestine than that of the control group. The number of MMC in the duodenum increased gradually after the infection and reached a peak on day 35 p.i. Thereafter, the number of MMC continued to decrease at a slow pace. The kinetics of MMC responses in the upper and lower jejunum were similar to that of the duodenum, but the number of MMC in the jejunum was lower. The worm recovery rate decreased with respect to time of which it was markedly reduced on day 49 and 56 p.i. The duration in which a high number of MMC appeared was similar to that in which a low rate in worm recovery was recorded. These results indicate that intestinal mastocytosis may play an important role in the expulsion of E. hortense.     

Echinostoma hortense and Heterophyid Metacercariae Encysted in Yellowfin Goby, Acanthogobius flavimanus, from Shinan-gun and Muan-gun (Jeollanam-do), Korea

Fishborne trematode (FBT) metacercariae were investigated in yellowfin goby, Acanthogobius flavimanus, collected from Shinan-gun and Muan-gun, Jeollanam-do (province), Korea. All collected fishes were examined using the artificial digestion method. In all of 15 gobies from Aphae-myeon in Shinan-gun, metacercariae of Stictodora spp. (334 metacercariae/fish), Heterophyes nocens (153/fish), and Heterophyopsis continua (20/fish) were detected. In 2 of 14 gobies from Jido-myeon in Shinan-gun, 8 Echinostoma hortense metacercariae in total were detected. In 15 gobies from Haeje-myeon in Muan-gun, the metacercariae of H. continua were found in 100%, Stictodora spp. in 86.7%, and H. nocens in 6.7% of fish examined. The average numbers of metacercariae per infected fish were 23.3 (H. continua), 416.0 (Stictodora spp.), and 2.0 (H. nocens), respectively. The metacercariae of E. hortense found in gobies were elliptical, with 150 × 138 µm in average size, and had 27 collar spines on the head crown. The above results suggest that yellowfin gobies from 2 localities may be the potential infection sources of FBT. Moreover, it is proved for the first time that the yellowfin goby, A. flavimanus, acts as a second intermediate host for E. hortense.     

Zoonotic Echinostome Infections in Free-Grazing Ducks in Thailand

Free-grazing ducks play a major role in the rural economy of Eastern Asia in the form of egg and meat production. In Thailand, the geographical location, tropical climate conditions and wetland areas of the country are suitable for their husbandry. These environmental factors also favor growth, multiplication, development, survival, and spread of duck parasites. In this study, a total of 90 free-grazing ducks from northern, central, and northeastern regions of Thailand were examined for intestinal helminth parasites, with special emphasis on zoonotic echinostomes. Of these, 51 (56.7%) were infected by one or more species of zoonotic echinostomes, Echinostoma revolutum, Echinoparyphium recurvatum, and Hypoderaeum conoideum. Echinostomes found were identified using morphological criteria when possible. ITS2 sequences were used to identify juvenile and incomplete worms. The prevalence of infection was relatively high in each region, namely, north, central, and northeast region was 63.2%, 54.5%, and 55.3%, respectively. The intensity of infection ranged up to 49 worms/infected duck. Free-grazing ducks clearly play an important role in the life cycle maintenance, spread, and transmission of these medically important echinostomes in Thailand.     

Tupaia Belangeri as an Experimental Animal Model for Viral Infection

Tupaias, or tree shrews, are small mammals that are similar in appearance to squirrels. The morphological and behavioral characteristics of the group have been extensively characterized, and despite previously being classified as primates, recent studies have placed the group in its own family, the Tupaiidae. Genomic analysis has revealed that the genus Tupaia is closer to humans than it is to rodents. In addition, tupaias are susceptible to hepatitis B virus and hepatitis C virus. The only other experimental animal that has been demonstrated to be sensitive to both of these viruses is the chimpanzee, but restrictions on animal testing have meant that experiments using chimpanzees have become almost impossible. Consequently, the development of the tupaia for use as an animal infection model could become a powerful tool for hepatitis virus research and in preclinical studies on drug development.     

A Case of Fasciola hepatica Infection Mimicking Cholangiocarcinoma and ITS-1 Sequencing of the Worm

Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.     

Postmetacercarial changes in Echinostoma caproni maintained in a defined medium plus calf serum

The present study examined postmetacercarial changes in the excysted metacercariae of Echinostoma caproni maintained in the defined medium Mixture 199 plus 20% calf serum for 7 days at 41 degrees C. The gas phase was atmospheric air. Each culture was inoculated with 25 excysted metacerariae. Cultures were maintained upright in closed 15 ml plastic centrifuge tubes each containing 10 ml of medium plus 200 units of penicillin/ml and 200 micrograms of streptomycin/ml. By 4 days in culture, most metacercariae had voided their excretory concretions. Organisms were clumped or solitary at the bottom of the cultures. Many organisms showed flaring of the oral collar and extension of both the collar and tegumentary spines. By 4 days in culture, posterior protuberances or bumps were noted on many of the organisms and some organisms showed abnormal vesicular growths or blebs at their posterior ends. Some mortality was noted in culture by day 5, but most organisms were still alive when the cultures were terminated on day 7.     

Echinostome Flukes Receovered from Humans in Khammouane Province, Lao PDR

Echinostome eggs were detected in 22 (1.1%) of 2,074 people residing in riparian villages along the Mekong River in Khammouane Province, Lao PDR. In order to recover the adult flukes, 9 persons were treated with praziquantel and purged with magnesium salts. A total of 55 echinostome specimens (4 species) were recovered from the 9 persons. Echinostoma revolutum (8 specimens) was recovered from 3 persons, Artyfechinostomum malayanum (8 specimens) was from 2 persons, Echinochasmus japonicus (33 specimens) was from 7 persons, and Euparyphium sp. (6 specimens) was from 1 person. In Lao PDR, only human infections with E. japonicus were previously known. Therefore, the present study describes human infections with E. revolutum, A. malayanum, and Euparyphium sp. for the first time in Lao PDR. These results indicate that the surveyed villages of Khammouane Province, Lao PDR are low-grade endemic foci of echinostomiasis.     

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.