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Expression and cross-species reactivity of fatty acid-binding protein of Clonorchis sinensis.

Clonorchis sinensis is a Chinese liver fluke that chronically resides in the biliary tract. The fatty acid-binding protein (FABP) is known to play an important role in the intracellular transport of long-chain fatty acids that are obtained by the fluke from the host. Although FABP has stimulated considerable interest as a vaccine target candidate, the nature of FABP from C. sinensis (CsFABP) remains unclear. In this paper, we describe the cloning and expression of recombinant FABP and immune cross-reaction by Western blot analysis. Sequence analysis revealed that the CsFABP cDNA contained a single open reading frame (ORF) coding for 134 amino acids with an estimated molecular mass of a 15.2 kDa. The DNA sequence of CsFABP cDNA showed significant homology to schistosome cytosolic FABPs, with a 49% amino acid sequence identity and 89% similarity to Schistosoma japonicum. This DNA also showed a high sequence similarity at the amino acid level to S. mansoni (Sm14; 83%) and Fasciola hepatica (80%). The CsFABP cDNA was cloned into expression vector pET28a, expressed in Escherichia coli and the recombinant protein purified by affinity chromatography. The recombinant CsFABP was cross-reacted with sera obtained from patients with fascioliasis and paragonimiasis. These results suggest that CsFABP may be useful as a vaccine for clonorchiasis.

Amino Acid Sequence↗

[Studies On The Experimental Mode Of Infections Of Clonorchis Sinensis: II. Experimental Infection Of Clonorchis Sinensis Cercaria To Fry Of Fresh Water Fishes]

Cercariae of Clonorchis sinensis were attempted to penetrate into various fry of fresh water fishes in order to examine the conditions of infection and the further development in the fish. The following conclusions were made. 1. Cercariae of C. sinensis penetrated into the muscle of the fry of Pseudorasbora parva were found to be able to develop. 2. Cercariae penetrated into the fry of Pseudorasbora parva migrated into the organs of the fry to kill their host. 3. From each dead individual of P. parva, more than 10 larvae of C. sinensis could be discovered. 4. Cercariae attached on the Cyprinus carpio, Carassius and gold fish were almost killed before penetration into the body and even one or two cercariae penetrated were also killed as time passed. 5. Cercariae of C. sinensis infected on the fry of Cyprinus Carpio, Carassius auratus and gold fish could not survive more than 60 days. 6. Cercariae penetrated into Cyprinus carpio and Carassius auratus could not develop into complete metacercariae even after 3O days. 7. Cercariae of Clonorchis sinensis penetrated into the fry of Aplocheilus latipes were killed within 10 hours and the cercariae attached on the caudal fin were destroyed in 2 hours. 8. It was found that no cercariae of Clonorchis sinensis could penetrate into the body of the fry of Misgurnus anguillicaudatus.

Journal Article↗

CT of Clonorchis Sinensis pancreatitis.

Clonorchis Sinensis infestation can involve the pancreatic duct and induce attacks of acute pancreatitis. Ct findings in a case of Clonorchis Sinensis pancreatitis revealed significant enlargement of the pancreas with compression and obstruction of the common duct. Medical treatment leads to resolution of the clinical and CT findings. The patient experienced a second episode of pancreatitis 3 yr later. CT examination documented the development of chronic pancreatitis with an acute exacerbation.

Clonorchiasis↗

Clonorchis sinensis in Kyungpook Province, Korea: 1. Distribution and demonstration of the cercaria of Clonorchis sinensis from snail, Parafossarulus manchouricus Bourgigant.

Studies were conducted in rivers in Kyungpook Province, Korea from July 1970 to September 1974 to determine the stations where Parafossarulus manchouricus, the intermediate snail host of Clonorchis sinensis, are abundant, and the infection rates of these snails with the cercaria of Clonorchis sinensis. Snail collections were made during four consecutive summers(July to September in each year). Ten snail habitats were found. The population density of the snails per square meter of the river bottom ranged from 25 to 250. The highest population of snails was encountered at the Dong-chon station. Cercaria studies by means of the immersing and crushing technics indicated surprisingly low rate of Clonorchis sinensis infection among the snails examined. The proportion of infected snails in Kyungpook Province was 0.8 per thousand of all snails examined. Results indicated that the Parafossarulus manchouricus exists in limited areas within the rivers under study and the infection of the snail with the cercaria of Clonorchis sinensis is very low.

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[Experimental studies on the second intermediate hosts of Clonorchis sinensis IV. Observations on the fate of metacercariae of Clonorchis sinensis in the fish host, Cultriculus eigenmanni]

In order to judge appropriately the suitability as the second intermediate host of Clonorchis sinensis, the present experiments were proposed to survey the metacercaria of C. sinensis in the fish host, Cultriculus eigenmanni, in the field, and to observe, by experimental infection of the fish with Clonorchis cercaria, the penetrating ability of the cercaria, maturity, the process of degeneration and extinction, and infectivity of the metacercaria. The following is a brief summary of the leading facts gained through the experiments. All the ten fish (mean body weight 13.4 +/- 3.43g) which were caught in Ok-ku irrigation reservoir were infected with the metacercariae and the average number of the metacercariae found in the fish body/gram was 4.55. The fish was easily subject to invasion of the cercaria, and the examination of the cercariae after having the fish infected disclosed that 24 hours after the infection, all of the cercariae formed their cysts in muscle and the metacercariae kept growing, that 7 days later the metacercariae were found folding their bodies twice, and that in 15 days the characteristic structure of the metacercariae was complete and made a vigorous rotary movement intermittently. In the case of control fish, Pseudorasbora parva, both penetrating ability of the cercariae and the infective degree of the metacercariae to control host were stronger as compared with the fish host because that the control fish had higher suceptibility to C. sinensis. Afterwards, the metacercariae in the fish host came to a state of maturity and beyond this stage some metacercariae started the process of degeneration and extinction in as early as 30 days, and as more days elapsed, mortality of the metacercariae was not uniformly increased but difference of mortality appeared to be according to the individual of fish host. That is, in 390, 540 and 586 days, all of the metacercariae in the fish hosts became dead, meanwhile almost all of those found in the fish host of 596 days after the infection survived (mortality; 8.51 percent). Percentage of orally administered metacercariae developing to maturity in rabbit is 10.46 percent in the last case. As the days were progressed the shapes of the metacercariae in the fish host tended to change from ellipsoid to almost round shapes, the length became shorter while the width was wider. The results shown above revealed that C. eigenmanni was, though it could not be proper host than P. parva, suitable as the second intermediate host in addition to our previous observation results.

Journal Article↗

Clonorchis Sinensis In Kyungpook Province, Korea: 3. Changing Pattern Of Clonorchis Sinensis Infection Among Inhabitants.

This study was undertaken to evaluate the effects of health education and anti-Clonorchis campaign conducted in Kyungpook Province and to compare the prevalence of Clonorchis sinensis among the residents in the same areas for the period 1964-1976. Both surveys were made on all age groups of residents ranging from 0 to over 60 years of age. They were examined using primarily intradermal tests with Clonorchis and Paragonimus antigens and followed by stool examinations. The definitive diagnosis of clonorchiasis was made on the recovery of Clonorchis eggs from the stools. A marked reduction in the prevalence was encountered in the youngest age group, but there is no significant difference in the older age groups. The overall prevalence rate for clonorchiasis was reduced from 27.7 to 19.6 per cent in a period of 10 years. It is found that clonorchiasis is less prevalent in Kyungpook Province than it was a decade ago and it continued to decline.

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Identification of IgE-reacting Clonorchis sinensis antigens.

Clonorchis sinensis is a liver fluke and it is the most prevalent human parasite in Korea at present. The parasite infection induces immune responses, characteristically an increased production of parasite-specific IgE in the host. Major IgE-reacting C. sinensis antigens in infected humans have been protein bands with MWs of 15, 28, 37, 45, 51, 56, 62, 66, 74, 97 and 160 KD identified by immunoblot analysis. Individual variations of the IgE binding pattern to C. sinensis antigens have also been documented. Using immune BALB/c mouse sera, IgE-reacting protein bands have been visualized with MWs of 28, 74, 86, 160 and several > 200 KD. One of the most strongly reacted C. sinensis antigenic proteins with a molecular weight of 28 KD was purified by gel filtration and preparative electrophoresis. Using a monoclonal antibody produced against the antigenic protein, the protein was localized in the parasite's intestine, and also found to be contained in excretory-secretory antigens.

Animals↗

Clonorchis sinensis in Kyungpook province, Korea: 2. Demonstration of metacercaria of Clonorchis sinensis from fresh-water fish.

From March 1973 to September 1974, fresh-water fishes were collected in rivers in Kyungpook Province, Korea, using three collecting technics; throwing a cast-net, fishing and using a glass bowl containing crushed oil-cake as a bait. Twenty-one species of the fish caught in the rivers in Kyungpook Province were examined for the presence of the metacercaria of Clonorchis sinensis. Of these, 10 speciec of the fish harbored the encysted larvae. The most frequently infected one was Pseudorasbora parva 92.3 per cent, followed by Pseudogobio esocinus 90.4 per cent, Sarcocheilichthys sinensis 87.7, Hemibarbus labeo 87.2, and Pungtungia herzi 60.8 per cent, in decreasing order. The less frequently infected were Erythroculter erythropterus 13.0, Acanthorhodeus taenianalis 7.7, Paracheilognathus rhombea 5.9 and Acheilognathus limbata 4.2 per cent. In the intensity of infection with the larvae, Pseudorasbora parva was most heavily infected and the mean number of metacercaria per ml of flesh was 51.4, Pungtungia herzi, Gnathopogon atromaculatus, Pseudogobio esocinus, Sarcocheilichthys sinensis and Homibarbus labeo were infected intermediately (Their mean number varied 10.3 to 19.5). Whereas, Paracheilognathus rhombea, Acheilognathus limbata and Acanthorbodeus taenianalis were infected with a few cysts.

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Lipofuscin Pigment In Adult Clonorchis Sinensis.

Adult Clonorchis sinensis from infected human cases may have black pigment in their parenchyme. In present paper, the nature of the pigment was investigated. The pigment distributed in cytoplasm of parenchymal cells of the worms. Histochemically the pigment was positive by periodic acid-Schiff, Ziehl-Neelsen, and lipofuscin stainings. The pigment particle contained many, various sized membrane structure as well as electron dense granules when observed by electron microscopy. Staining characteristics of the black pigment, together with electron microscopic findings, strongly indicated that the pigment was lipofuscin.

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[Studies on the experimental mode of infections of Clonorchis sinensis: III. Studies on the wormicidal effect of external mucous substance of some fresh water fish on the larva of Clonorchis sinensis]

The author reported in the previous pager on his observation of sudden death of Clonorchis sinensis cercaria before penetration into the body of fry such as Cyprinus carpio and Carassius auratus. And in these studies, the reason behind the fact was investigated and it was found that the mucous substances on the surface of a certain fishes have a strong wormicidal effect to the larva of C. sinensis. 1. When cercariae of C. sinensis were immersed into the mucous substance of gold fish for three minutes 36 seconds to 4 minutes 20 seconds, into that of Aplocheilus latipes for 4 minutes 30 seconds to 5 minutes 20 seconds, into that of Cyprinus carpio for 5 minutes 15 seconds to 6 minutes l6 seconds, and into that of Carassius auratus for 5 minutes 20 seconds to 7 minutes 30 seconds, it was observed that they were all killed. 2. No wormicidal effect against C. sinensis could be observed with the mucous substance obtained from Pseudorasbora parva, Misgurnus anguillicaudatus and Ophicephalus argus. 3. No wormicidal effect against Metagonimus sp. cercaria is observed with the mucous substance obtained from Cyprinus carpio, Carassius auratus. 4. The wormicidal effect of the mucous substances obtained from gold fish was destroyed when heated over 60 degrees C. 5. The movements of metacercaria of C. sinensis stopped after 10 minutes application of the mucous substance obtained from Cyprinus carpio and they were killed within 30 minutes. 6. When the adult worms collected from the bile duct of experimentally infected rabbits for 20 to 60 days, were immersed into mucous substance from Cyprinus carpio, they were killed between 25 minutes and 35 minutes. 7. No wormicidal effect could be observed with the mucous substance obtained from dead Cyprinus carpio, Carassius auratus, gold fish, when applied to the adult worms and larvae of C. sinensis.

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Molecular cloning and characterization of a novel lactate dehydrogenase gene from Clonorchis sinensis.

From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel lactate dehydrogenase (LDH) gene which encoded a putative protein with a predicted molecular weight of 35.6 kDa. The optimum pH and temperature for the enzyme were 7.5 and 50 degrees C in the pyruvate reduction while 11 and 80 degrees C in the lactate oxidation reaction, respectively. CsLDH showed no substrate inhibition by high lactate and NAD(+) concentration, and the optimal pyruvate and optimal NADH concentrations were 10 and 0.5 mmol/l, respectively. The relative activities of these 2-oxocarboxylic acids were pyruvic acid>2-ketobutyrate>oxalacetic acid>alpha-ketoglutaric acid>phenylpyruvate. The cofactor 3-acetylpyridine adenine dinucleotide was much more effective than NAD(+). The cofactor analogs in which the nicotinamide ring is replaced by 3-pyridinealdehyde were lower activity cofactors, while the nicotinamide ring is replaced by nicotinic acid or thionicotinamide which is not a cofactor to CsLDH. The succinic acid and malic acid are not substrates of CsLDH. Cu(2+), Fe(2+), and Zn(2+) greatly inhibited the CsLDH activity both in the direction of pyruvate reduction and in the direction of lactate oxidation. The inhibition of CsLDH by gossypol may make gossypol a potential therapy drug or a lead compound for C. sinensis. Accordingly, the CsLDH may be a novel potential drug target.

Amino Acid Sequence↗

Molecular cloning and immunological characterization of phosphoglycerate kinase from Clonorchis sinensis.

The parasite Clonorchis sinensis was determined to utilize a large amount of external glucose to carry its energy metabolism. Phosphoglycerate kinase (PGK), a glycolytic enzyme, found in many parasites, has been identified as one of the candidate molecules distinguished from human counterparts for vaccine and drug developments. A cDNA clone purified by screening a C. sinensis cDNA library using a heterologous cDNA probe encoded a putative peptide of 415 amino acids with over 60% identities with PGKs from a number of animals. The putative peptides revealed domains corresponding to 12 beta-sheets and inner loops forming a substrate-binding cleft of animal PGKs. The gene product was overexpressed in Escherichia coli and showed a PGK-like enzyme activity. A polyclonal antibody raised against the recombinant C. sinensis PGK was specific to native C. sinensis PGK and localized it to the muscular tissue and tegument of the adult flukes. The C. sinensis PGK elicited antibodies in C. sinensis-infected rabbits. Therefore, it is proposed that C. sinensis PGK could be used as an immunoreagent in the serodiagnosis for clonorchiasis.

Amino Acid Sequence↗

[Experimental studies on the second intermediate hosts of Clonorchis sinensis: III. Observations on the relationship between clavate cells of epidermis and infectivity of metacercariae of Clonorchis sinensis in fresh-water fish]

In order to know relationship between the clavate cells of epidermis and infectivity of metacercariae of C. sinensis. histological investigation of epidermis was carried out on the 26 species of fresh-water fish collected from the main rivers in Korea. Results were summarized as it follows: 1. In general clavate cell had a single nucleus at its center. However it was observed in Liobagrus mediadiposalis and Pelteobagrus fuluidraco that clavate cell had two nuclei with and independent so-called light halo of Oxner and in some cases had none of them. 2. In following species; Misgurnus anguillicaudatus, Cobitis taenia, Liobagrus mediadiposalis, Pelteobagrus fuluidraco, Parasilurus asotus and Anguilla japonica, scales were not detected or buried in the dermis layer, and the epidermis was mostly consisted of clavate cells. Among these species, it was observed that no metacercariae were found. 3. In case of Cyprinus carpio, Carassius carassius (crusian carp and golden crusian carp), Sarcocheilichthys czerskii, Pungtungia herzi and Coreoleuciscus splendius, scales were exposed over the epidermis, in which clavate cells were abundantly found. In these species, a few of the metacercariae were detected. 4. On the other hand, no clavate cells were observed in Pseudorasbora parva, Zacco platypus, Microphysogobio koreensis, Gnathopogon majimae, Gnathopogon coreanus, Acheilognathus signifer, Acheilognathus yamatsutae, Cultriculus eigenmanni, Coreoperca herzi, Pseudoperilampus uyekii, Pseudoperilampus notatus and Pseudogobio esocinus. These fishes with exposed scales on the epidermis were well known to serve as the second intermediate hosts of C. sinensis. From the above results obtained, it was suggested that the existance of clavate cells in the epidermis of the fresh-water fish seems to be correlated with the infectivity of the metacercariae of C. sinensis.

Journal Article↗

Molecular cloning and characterization of a mu-class glutathione S-transferase from Clonorchis sinensis.

In biliary passages, Clonorchis sinensis causes epithelial hyperplasia and is assumed to promote carcinogenesis. Glutathione S-transferase (GST) is an antioxidant enzyme involved in phase II defense in trematodes. A clone (pcsGSTM1) encoding a GST was identified by screening a C. sinensis cDNA library with a PCR-synthesized cDNA probe. The predicted amino acid sequence encoded by pcsGSTM1 cDNA had a high degree of sequence identity and folding topology similar to the mu-class GSTs. The estimated molecular mass of the protein, 26 kDa, was consistent with an expression by pcsGSTM1 cDNA. The bacterially expressed recombinant csGSTM1 protein possessed an enzymatic GST activity and conjugated GSH to reactive carbonyls of lipid peroxidation. The recombinant csGSTM1 protein did not share antigenic epitope(s) with GSTs of Fasciola hepatica, Paragonimus westermani and Schistosoma japonicum. The csGSTM1 was identified to a mu-class GST in C. sinensis.

Amino Acid Sequence↗

Long-term storage of Clonorchis sinensis metacercariae in vitro.

Clonorchis sinensis metacercariae must often be stored for several months to meet laboratory requirements. In the present study, the authors evaluated morphological changes and infectivities of metacercariae kept in different storage solutions for 1, 3, and 6 months, i.e., PBS (phosphate buffered saline), PBS + antibiotics, PBS + iodoacetic acid (IAA), and PBS + antibiotics + IAA. Metacercariae stored in PBS containing antibiotics showed significantly higher survival rates and infectivities than those stored in PBS alone after 3 or 6 months. The metacercariae that had been stored for 6 months in solutions that were refreshed monthly had also significantly higher survival and infection rates than those stored in nonrefreshed solutions. Moreover, the addition of IAA, an inhibitor of cysteine proteinase, to these storage solutions reduced the survival and infectivity. The results of the present study confirm that the viabilities of C. sinensis metacercariae are best maintained in storage solutions containing antibiotics that are regularly refreshed.

Animals↗

The longevity of Clonorchis sinensis.

The persistence of Clonorchis sinensis infestation of the bile duct for a period of at least 26 years without neoplasia supervening is reported in a Chinese immigrant to Australia.

Aged↗