Fibricola Seoulensis Research Articles

Neodiplostomum seoulensis n. comb. (Trematoda: Neodiplostomidae)

Fibricola seoulensis Seo, Rim, and Lee, 1964 was described from rats in Seoul, Korea and was later found to be a natural parasite in the intestine of humans. At the time this species was described, the classification of this and other strigeiform flukes was based largely on the phylogeny of the host; those adult worms parasitic in birds were placed in Neodiplostomum and those parasitic in mammals were placed in Fibricola. A recent analysis of this group produced a classification that was based on the phylogeny of the worms themselves and suggested that certain metacercarial and adult characters were more important in determining the phylogenetic position of these worms than was the host. In light of the new classification, we examined newly collected metacercariae and re-examined fixed adults of F. seoulensis and found their characters to be more compatible with the genus Neodiplostomum. The characters included a neodiplostomulum metacercaria with 4 transverse commissures and an adult stage with symmetrical anterior testis and vitellaria in both the fore- and hindbody. Therefore, it is recommended that F. seoulensis be recombined as Neodiplostomum seoulensis.

Worm recovery rate and small intestinal lesions of albino rats coinfected with Fibricola seoulensis and Metagonimus yokogawai

Worm recovery rates and pathologic changes in small intestine of albino rats were observed after concurrent and challenge infections with metacercariae (MC) of Fibricola seoulensis and Metagonimus yokogawai, and compared with those of single infection groups. Albino rats in concurrent infection group were killed 20 days after feeding with 1,000 MC of each fluke. Rats in challenge infection group were fed with 1,000 MC of F. seoulensis and challenged by 1,000 MC of M. yokogawai 10 days after primary infection, then killed 10 days thereafter. In concurrent infection group, mean number of F. seoulensis and M. yokogawai recovered, 250 and 118 respectively, were similar to those of single infection groups. However, more flukes were collected from the duodenum and less flukes were from the ileum than from single infection group. In challenge infection group, the recovery rate of F. seoulensis was similar to that of single infection group and the distribution of the flukes was similar to that of concurrent infection group. Mean number of M. yokogawai, 69, was significantly lower than that of single infection group. Its distribution, however, extended to the duodenum and most of the flukes were recovered from the jejunum. In concurrent infection group, villi of the duodenum were more markedly thickened, fused and shortened than those in F. seoulensis single infection group. The crypt epithelium appeared to be hyperplastic and inflammatory cell infiltration into the villous stroma was mild. Villous atrophy in the jejunum and ileum was milder than in M. yokogawai single infection group. In challenge infection group, the findings were similar to those of concurrent infection group.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and isozyme patterns of phosphatase in Fibricola seoulensis

The present study was carried out to investigate the localization and isozyme patterns of acid phosphatase and alkaline phosphatase in metacercariae and in adults of F. seoulensis by enzyme-histochemistry method and electrophoresis. Acid phosphatase showed a strong activity at pH 5 in the intestinal caecum of adults, but showed no reactions in the non-substrate control and in the inhibitor-treated control. Alkaline phosphatase showed a strong activity at pH 8 in the intestinal caecum and the tribocytic organ of adults, and in the intestinal caecum and in the genital anlagen of metacercariae. In non-denature PAGE, ten bands of protein fraction from the extracts of metacercariae and twenty-two bands from adults were detected. In denature PAGE, two protein bands having molecular weights of 192 kDa and 123 kDa were detected in the metacercariae, but absent from adult stage. In adults, protein fractions of 27.5 kDa, 24.5 kDa, 21.4 kDa, 18 kDa, 16 kDa and 15 kDa were detected. In non-denature PAGE, isozymes of acid phosphatase showed the most strong activity at pH 5, whereas no activity was shown at pH 2 and pH 7. One isozyme band (62 kDa) of AcPase was recognized in metacercariae and 4 isozyme bands (95 kDa, 85 kDa, 73 kDa and 62 kDa) in adults.

Transmission electron microscopic ultrastructures of the male germinal cells of Fibricola seoulensis

A transmission electron microscopic study was performed to observe the ultrastructures of the male germinal cells and spermatozoa of Fibricola seoulensis. Spermatogonia were found in the periphery of the testis and characterized by large nuclei and comparatively little cytoplasms. Spermatocytes contained an oval to spherical nucleus. Their nuclear volume was little larger in comparative to that of cytoplasm, and the chromatin was comparatively little. The early spermatids were characterized by a great amount of cytoplasm, and numerous mitochondria encircled the nucleus. In a more advanced spermatids the electron-dense strands of chromatin appeared in the nucleus, and a pair of rootlet of the axoneme and a microtubule-organizing center (MTOC) were observed near the nucleus. The sectioned spermatozoa were found in the testis and the seminal vesicle. Their cross sectional views were divided into 6 types when they were distinguished on the basis of the morphology and components. The spermatozoa of F. seoulensis showed two flagella of 9+1 type axoneme.

Activities of acid phosphatase and non-specific esterase are present in the tribocytic organ and the caecum of Fibricola seoulensis.

In order to know the enzyme activities of Fibricola seoulensis, an intestinal trematode of human and rodent in Korea, the enzyme histochemical method is applied. Activities of acid phosphatase (E.C.3.1.3.2) and non-specific esterase (E.C.3.1.1) were present in microvilli and glandular cells of tribocytic organ and the epithelium of the caecum.

The nervous system of Fibricola seoulensis by acetylcholinesterase histochemistry

F. seoulensis were obtained from artificially infected albino rats at 3, 4, 5, 6, 7 days after infection. The worms and metacercariae were washed in physiological saline solution, and fixed with 10% neutral formalin. The acetylcholinesterase (AchE) stained by enzyme histochemistry using acetylthiocholine iodide as substrate. Eserine, iso-OMPA and BW284C51 were used as inhibitors of AchE. The nervous system consists of three pairs longitudinal nerve trunks interconnected with excretory plexus in posterior half, and pharynx and oral sucker in anterior half of metacercariae and adults. The longitudinal nerve trunks are interconnected with transverse commissures and numerous circular commissures. Considerable numbers of circular commissures are interconnected with longitudinal nerve trunks lying on the surface of the worms. At each stage of juvenile worms, AchE and nonspecific cholinesterase activities were observed in the oral sucker, ventral sucker, pharynx and nerve system. Isozymes of AchE in F. seoulensis were separated into the two bands, 69 kDa and 132 kDa. The major band was 69 kDa.

Transmission electron microscopic findings of the tribocytic organ of Fibricola seoulensis.

We observed ultra-structure of the tribocytic organ of Fibricola seoulensis with transmission electron microscope. Microvilli are observed on the surface of the tribocytic organ. Below the muscle layer, we can find three types of cells. Type I cell has electron lucent cytoplasm with a few granules, while type II cell shows electron dense cytoplasm and the particulate granules. Type III cell's electron dense cytoplasm possesses many granules. Of the above three cells, Type I and II cells are believed as tegumental cells. Type III cells are thought as glandular cells specific to the tribocytic organ. This finding can also explain the secretory function of the tribocytic organ of F. seoulensis.

Echinostoma revolutum and Echinoparyphium recurvatum recovered from house rats in Yangyang-gun, Kangwon-do

During an investigation on intestinal flukes of house rats in Yangyang-gun, Kangwon-do, a total of 6 species of trematodes belonging to 3 families; Echinostomatidae (Echinostoma hortense, E. cinetorchis, E. revolutum and Echinoparyphium recurvatum), Diplostomidae (Fibricola seoulensis) and Plagiorchiidae (Plagiorchis muris), were recovered from two adult rats. E. revolutum and E. recurvatum were new trematode faunae of rats in Korea. E. revolutum had an elongated body, 5.3-6.0 mm long and 1.0-1.3 mm wide. The total number of collar spines was 35-37 including 5 end group ones on each ventral corner. Its coiled uterus contained numerous eggs. E. recurvatum also had an elongated body, 3.5-4.7 mm long and 0.50-0.65 mm wide. It had total 45 collar spines including 4 end group ones. The uterus was short with only a few eggs. It has been first confirmed by this study that E. revolutum and E. recurvatum are indigenously distributed among house rats in Korea.

Histochemical findings of the tribocytic organ and tegument of Fibricola seoulensis

The tribocytic organ and tegument of Fibricola seoulensis were examined histochemically for the detection of carbohydrates, mucosubstances, amyloid, collagen and alkaline phosphatase. The surface, secretes, gland cells of the tribocytic organ, and the tegument of the worms were positive to periodic acid Schiff (PAS) and PAS with diastase stain but negative to other stains. It was inferred that the tribocytic organ and tegument of F. seoulensis comprise neutral mucopolysaccharides, which may take a protective role against host enzymes. The surface and secretes of the tribocytic organ, and the tegument of the worms were also positive to double bridge PAP for alkaline phosphatase. This fact suggests that they may play a role as both self protective and host tissue lytic functions.

Observation of mucosal pathology after praziquantel treatment in experimental Fibricola seoulensis infection in rats

It is well known that the duodenum of mice or rats infected with Fibricola seoulensis shows atrophy of villi (shortening, blunting, widening, fusion) and hyperplasia of crypts. This study was performed to observe healing process of these pathological changes after deworming with anthelmintic treatment. Albino rats infected each with 1,000 metacercariae of F. seoulensis were treated with single dose of 10 mg/kg praziquantel on day 15 post-infection. On day 1, 3, 5, 7, 15, 21 and 28 after the treatment, they were sacrificed and their duodenums were histopathologically studied. Control (uninfected) rats showed their normal finger-like projections of duodenal villi and well arranged crypts. In comparison, untreated (infected) controls revealed severe mucosal changes characteristic of villous atrophy and crypt hyperplasia in their duodenum. The damaged duodenal mucosa was found to restore its normal morphology after praziquantel treatment; until day 3 post-treatment the mucosa was severely atrophied; on day 5 long and slender villi sometimes appeared among the fused and stout ones; after day 15 the villi were in their normalizing process. From this experiment, it was shown that the mucosal changes in the duodenum of rats caused by F. seoulensis infection were completely reversible in 21-28 days after anthelmintic treatment.

Clinical and histopathological findings in mice heavily infected with Fibricola seoulensis

To observe the clinical course and intestinal histopathology in heavy infection of Fibricola seoulensis, an experimental study was performed in mice. Clinical, hematological and histopathological observation was done on 1, 3, 7 and 12 days after experimental infection with l,000 metacercariae. On the 11th day after infection, the mice began to die and all of the infected mice were dead by the 16th day. The infected mice showed gradual weight loss. Occult blood was detected after the 10th day. Diarrhea occurred after the 9th day and was recognized in all of the infected mice on the 12th day. Hemoglobin and mean corpuscular hemoglobin decreased significantly after the 12th day, and mean corpuscular hemoglobin concentration decreased in all infected mice. The histopathological changes were more marked in the duodenum than in the jejunum or ileum. Major changes were villous atrophy and crypt hyperplasia, with decreased villus/crypt ratio, inflammatory cell infiltration and stromal edema. The present results suggest that the cause of death of mice heavily infected with F. seoulensis should be malnutrition and severe fluid loss due to malabsorption, together with intestinal bleeding.

The life cycle and larval development of Fibricola seoulensis (Trematoda: Diplostomatidae).

The life cycle of Fibricola seoulensis was studied in the laboratory and in the field, with special interests in the larval developments within the eggs and in the intermediate hosts. The first emergence of miracidia after incubation of eggs in 26C water began on the ninth day. The miracidia, elongate and cylindrical shape, had epidermal plates in the formula of 6, 9, 4 and 3, with two pairs of flame cells and lateral processes. A kind of fresh water snail, Hippeutis (H.) cantori, was found to shed furcocercous cercariae from the 13th day after experimental challenge with miracidia while Physa acuta failed to shed. The same kind of snail collected from the field also shed the same cercariae. The cercariae were equipped with 2 pairs of penetration glands and 5 pairs of flame cells. The tadpoles of Rana nigromaculata were found susceptible to experimental infection with the cercariae. The same kind of tadpoles collected from various areas were also found naturally infected. The metacercariae in the tadpoles which were infected experimentally became infective to the definitive host in 21 days. The metacercariae were located free in the body cavity of tadpoles, and attained sexual maturity in rats in 7 days. The present study successfully followed the complete life cycle of F. seoulenis and found that it is possible to maintain the life cycle in the laboratory.

Egg laying capacity of Fibricola seoulensis in mice and rats.

The egg laying capacity of Fibricola seoulensis was observed in mice and rats. In mice, the mean number of eggs produced by an adult worm a day was 116 at the first week after infection, 123 at the 2nd week and 42 at the 3rd week. Thereafter, the capacity of about 20 eggs/worm/day was maintained up to one year period. In rats, it generally produced more eggs; 49 at 1 week, 250 at 2 weeks, 216 at 4 weeks, 327 at 6 months and 11 at one year after infection. It survived longer than a year in mice and rats.

A survey on intestinal parasites of soldiers in Korea

Total of 2,643 Korean soldiers were examined of their stool for parasitic infections by both cellophane thick smear and formalin-ether concentration techniques from August 1983 to December 1985. Out of them, 73.6% were free from any parasite, 22.6% were ova positive and 4.0% cyst positive. The ova positive rates by species were Ascaris lumbricoides 2.0%, Trichuris trichiura 13.0%, hookworm and Trichostrongylus orientalis 0.08% respectively, Clonorchis sinensis 7.6%, Metagonimus yokogawai 1.1%, Paragonimus westermani 0.08%, Echinostoma hortense 0.04%, Fibricola seoulensis 0.9%, Taenia sp. 0.3%, Hymenolepis nana 0.2% and H. diminuta 0.04%. Most of them (87.3%) were positive by a species, 11.9% by two and 0.8% by 3 species. The cyst positive rates were Entamoeba histolytica 0.6%, E. coli 1.4%, Endolimax nana 1.7%, Iodoameba butschlii 0.04% and Giardia lambla 0.9%. Among the cyst positives, 89.5% were positive by a species, 7.6% by two and 0.5% by 3 species. The intestinal parasite infections among the Korean soldiers decreased distinctly compared with previous data.

Ten human cases of Fibricola seoulensis infection and mixed one with Stellantchasmus and Metagonimus.

Ten soldiers of the Korean Army were found to have F. seoulensis ova in their faeces. These men were treated with praziquantel and the worms passed after purging were successfully collected in one case: 59 F. seoulensis, 68 Metagonimus and 2 Stellantchasmus falcatus were collected. 2 men were also infected with Clonorchis sinensis. All the cases of fibricoliasis had eaten raw or undercooked snake meat or frogs during survival training. All were egg-negative 2-6 months after treatment.

The effect of temperature and salinity on maturation and hatching of Fibricola seoulensis eggs.

This study was carried out to reveal the effect of temperature, salinity and aeration on maturation and hatching of Fibricola seoulensis eggs. The eggs were incubated and were observed daily for the appearance of eyespots and hatching. The results were summerized as follows: From 4-5 days after incubation in distilled water at 28C or at 11-26C, the eyespots began to appear and the rate of eggs with eyespots were over 90% in 28C on the 7th or 8th day. However, eyespots did not appear in 5-15C or 4C by the 18th day. The mature eggs began to hatch at the 8th day, and hatching rate 2 weeks after incubation was over 90% at 28C, but it was below 5% at 11-26C, and 0% at 5-15C and 4C. Aeration did not influence the appearance of eyespots nor hatching. In saline under 0.6%, the rates of eyespots were over 90% on the 7th day. The rate was 55.0% in 0.9% at 20 days, and 0% in 1.2%. The hatching rates in salines below 0.3% concentration were over 90% by 14 days of incubation. However, the rate decreased to 44% in 0.6% saline and to 0% over 0.9% salinity. The eggs incubated in the dark hatched in 12.5% on the 10th day, but hatching rate of maturation eggs increased to 85.7% within 2 hours after exposure to light. Above results demonstrated that the best temperature for maturation and hatching of F. seoulensis eggs was 28C, and the miracidia began to hatch at 8-9 days after incubation. In the field hatching and invasion into snails of the miracidia may occur from May to September in Korea. In salines under 0.3 percent concentration maturation and hatching were not influenced, but as salinity increased hatching was inhibited more than maturation was.

Growth and development of Fibricola seoulensis metacercariae in tadpoles.

In order to observe the growth and development of Fibricola seoulensis metacercariae, the tadpoles of Rana nigromagulata were experimentally infected with the cercariae. The metacercariae of various developmental stages were recovered from the tadpoles after 2 to 65 days of infection. They were prepared for morphological observation, and were given orally to mice to observe their infectivity. The following results were obtained. All of the tadpoles exposed to the cercariae were observed to harbour the larvae in their abdominal cavity. The young metacercariae of 2 days after infection were 121.1 micrometer long and 63.3 micrometer wide. They grow linearly for the first 14 days to be 262.0 micrometer long and 166.4 micrometer wide. Thereafter, no more growth recognized until 65 days. The larvae of 2 days old were similar with cercarial body and had 2 suckers, a pharynx, two ceca and a primordium of germ cells but no tribocytic organ. On the 8th day, they had tribocytic organ, and their morphology resembled that of mature metacercariae. The metacercariae younger than 10 days could not infect the mice. Only the metacercariae older than 14 days had infectivity. The recovery rate increased by the age of metacercariae from 19.0% in 14 days old to 70.0% in 40 days old. Above findings indicate that the tadpole is indispensible for metacercarial development and it needs at least 2 weeks for maturation. The tadpole is a pivotal host in the life cycle of F. seoulensis for connection between the snail and the frog.

A histopathological study on the intestine of mice and rats experimentally infected by Fibricola Seoulensis.

A histopathological study was carried out on the duodenum of mice and rats experimentally infected by F. seoulensis. Each mouse was infected wit 500 metacercariae and killed after 1, 2, 3 days, 1 and 2 weeks from infection. Each rat was given 1,000 metacercariae and was examined after 1, 2, and 4 weeks from infection. The duodenal tissue sections of mice and rats were stained with hematoxylin and eosin, and PAS stained for the rats of 1 week group. The pathological findings are summarized as below. There were no differences in mucosal findings between the mice and the rats, adn between the location of duodenum, 1 and 5 cm distal to the pylorus. Each worm embraced a villus exclusively with its foliate forebody which was inserted into the intervillous spaces. The fluke pinched villous epithelia using its oral and ventral suckers. The tribocytic organ destroyed the villous epithelia deeply up to the stroma after 3 days from infection. Apparent villous changes were observed in the mice after 3 days from infection. Villous changes were shortening, widening, blunting or fusion. The villous stroma showed edema, microscopic hemorrhage, capillary congestion, dilatation of lymphatics and inflammatory cell infiltration. The cells were lymphocytes, plasma cells, eosinophils and giant cells. Rarely submucosal and transmural inflammation was encountered.

A study on the infection status of tadpoles and frogs by the metacercariae of Fibricola seoulensis in Korea

A total of 220 tadpoles was captured in 6 areas and total 117 frogs, Rana nigromaculata, were collected in 11 areas in Korea. They were examined for their infecion status by the metacercariae of Fibricola seoulensis by peptic digestion technique and by histological observation with hematoxylin-eosin staining. This study was carried out from August 1983 to September 1984. Followings are the results. The tadpoles of R. nigromaculata were positive for the metacercariae from 3.3 percent to 100 percent by area. The number of metacercariae per infected tadpole ranged from 1 to 584, and the mean number per tadpole ranged from 7.6 to 221 by area. The metacercariae from 16 tadpoles were counted by the body portion. A great majority of the metacercariae was collected from abdominal cavity, 98.3 percent of 484 counted larvae. And 6(1.2 percent) larvae were from proximal tail and 2(0.4 percent) from trunk. Histological sections of tadpoles showed many metacercariae in abdominal cavity but none in other parts. The larvae were free in the spaces among intestinal loops or around primitive liver. A few larvae were in duct-like tissues near trunk wall. There was little infiltration of inflammatory cells. The metacercarial infection rates of frogs ranged from 0 percent to 100 percent by area. The larval burden was 1 to 470 by infected frogs, and mean number ranged from 1 to 175.6 by area. By above results, it is suggested that the cercariae of F. seoulensis may infect R. nigromaculata already in the stage of tadpole. Almost all of the metacercariae were concentrated in abdominal cavity of tadpoles. According to the infection status of frogs, this fluke is prevalent almost nation-widely in rice paddies in Korea.

Studies on intestinal trematodes in Korea XIX. Light and scanning electron microscopy of Fibricola seoulensis collected from albino rats treated with praziquantel.

An experimental study was performed to observe the in vivo effects of praziquantel on the light and scanning electron microscopic morphology of Fibricola seoulensis. The metacercariae were obtained from the snakes and 1,000 in each number was orally given to total 15 albino rats; 5 controls and 10 treatment group. Seven days later the 10 rats were treated with 10 mg/kg praziquantel and sacrificed 1~24 hours later to search for the worms from their small intestines. The major light microscopic changes in the drug-exposed worms were early contraction followed by relaxation of especially their forebody, vacuolization of the tegument and subtegumental parechymal layers, and narrowing of the intestinal lumens. The scanning electron microscopic findings were characterized by formation of numerous blebs followed by rupture and subsequent destruction of their whole tegument. These results show that the change in worm body is not confined to the tegument but extends to deeper parechymal portions and also occurs in their intestines. It is suggested that the drug uptake by the worms should be either through their tegument or through the digestive tract.