Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.     

Studies on the excystment of trematode metacercariae in vitro.

Excystment of metacercariae of Parorchis acanthus, Posthodiplostomoides leonensis, Posthodiplostomum sp. and Clinostomum tilapiae was found to be an active process initiated by the metacercariae when the correct physico-chemical conditions exist in the environment; but in the excystment of Posthodiplostomum nanum the metacercaria seems not to take part in the process. Temperature, osmotic pressure, pH and eH are factors which influence all the five species studied. Fresh ox bile was found not to be an absolute necessity for the excystment of the five species studied but its presence in the medium increased the rate of excystment of three species.     

Metamorphosis of the excretory system of Paragonimus ohirai (Trematoda), with special reference to its functional significance

The fine structure of the excretory system was studied in metacercariae and juveniles of Paragonimus ohirai. The former were in vitro excysted, and the latter were collected from the abdominal cavity of a rat 24 hours postinfection. The terminal organs of the excretory system were composed of a flame cell and the first cell of a tubule. In the excysted metacercaria, there was no space between these cells to allow the passage of fluid. This suggests that the terminal organs of P. ohirai may be inactive in this stage. The excretory bladder was formed of epithelial cells which contained numerous lipid droplets and a large amount of glycogen in the cytoplasm. The bladder of the metacercaria seems to function as a storage area for nutrients and other materials. These characteristics of the metacercaria are considered to be related to the enclosed conditions created by encystment. Excretory organs had undergone cytomorphosis in the juveniles and appear to possess active excretory function. The periflagellar space in the terminal organs was formed, and lipids and concretions were excreted from a thin layer of the excretory epithelia into the lumen.     

Blastogenesis of splenic lymphocytes to specific antigens and PHA in Paragonimus westermani infected mice]

Paragonimus westermani is a common fluke in Korea. The present study aimed to observe the cell mediated immune response in experimental paragonimiasis of mice. The mouse (BALB/c) was orally inoculated with 40 metacercariae of P. westermani from Cambaroides similis. During the infection (1, 2, 4, 6 weeks) of mouse, blastogenic response of splenic lymphocytes to P. westermani adult antigen, metacercaria antigen, and PHA were observed. Sera from infected and noninfected mice added to normal mouse splenic lymphocytes with or without PHA. The blastogenic response of splenic lymphocytes to PHA was reduced after 1 week of infection. However after 6 weeks of infection, the response was restored to the control level. The blastogenic response of splenic lymphocytes to P. westermani adult or metacercaria antigen increased significantly on 1 week after infection, and maintained up to 6 weeks after infection. The response of non-infected mice was suppressed by addition of the infected mouse serum. The present results suggested that cellular immunity was involved in P. westermani infected mice and that P. westermani anti-serum inhibited proliferation of T lymphocytes.     

Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

Histochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.     

In vitro excystment of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae)

1986. In vitro excystrnent of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae). International Journal for Parasitology 16: 641–645. An optimal hatching success of Plagiorchis species 1 metacercariae (100% excystment, active metacercariae, mean hatching speed 2–10 min, lowest variance of the mean speed) was observed after pretreatment in an HCl-pepsin solution at pH 2.0 and 42°C for 60–70 min, and incubation in a hatching medium at 42 °C and pH 7.3–8.0 with a bile salt (Nacholate), NaHCO₃, and a reductant (cysteine with 100% N₂). The minimum conditions for nearly 100% excystment with lower hatching speeds and higher variances were the presence of NaHCO₃, an oxygen concentration reduced to about 3% in the gas phase, pH> 7.3 and a temperature near 30°C if Na-cholate was absent, or in the presence of the bile salt, a phosphate buffer at pH> 5.0 and room temperature only. Obviously some hatching factors acted interchangeably with compensation for missing stimuli by others. The effect of the bile salt was comparable with that of other surfactants. The metacercariae excysted in nonenzymatic media, which implies an active hatching mechanism.     

Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187

The involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

Antibody-dependent rat macrophage-mediated damage to the excysted metacercariae of Paragonimus westermani in vitro

An in vitro immune effector mechanism against the target excysted metacercariae of Paragonimus westermani was demonstrated in the rat system. Peritoneal exudate cells, mainly macrophages from normal rats, showed adherence to and killing of excysted metacercariae of P. westermani in the presence of complement-independent serum from rats infected with Paragonimus metacercariae. These reactions were specific for the excysted metacercariae, as tissue-migrating juvenile worms were not affected. Damage of excysted metacercariae of P. westermani due to antibody and macrophages was assessed by morphological observation, by cell adherence reaction and by the use of vital dyes. Trypan blue dye exclusion proved to be a reliable indicator of judging metacercarial viability. Electron microscopic studies demonstrated that macrophages reacted with fuzzy material on the tegumental surface and fine structures in the syncytium of the parasites. The tubular tunnels formed between the basement membrane and muscle layers of the damaged parasites were also noticeable. The relevance of these findings to cellular immunity in the early paragonimiasis was discussed.     

Infection status of Paragonimus westermani metacercariae in crayfish (Cambaroides similis) collected from Bogildo (Islet), Wando-gun, Chollanam-do, Korea

During the period from October 1996 to November 1998, the infection status of Paragonimus westermani metacercariae in freshwater crayfish (Cambaroides similis) collected from Bogildo (islet). Wando-gun, Chollanam-do, which is known for an endemic area of P. westermani in Korea, were examined. The average infection rate of Paragonimus metacercariae in crayfish was 88.6%, and mean number of metacercariae per infected crayfish was 30.2. This metacercarial density was the highest in the group of weight in 7.1-9.0 g. These results suggest that the natural life cycle of P. westermani is still well-preserved in Bogildo.     

A 27 kDa cysteine protease secreted by newly excysted Paragonimus westermani metacercariae induces superoxide anion production and degranulation of human eosinophils

Eosinophil degranulation plays a crucial role in tissue inflammatory reactions associated with helminth parasitic infections and allergic diseases. Paragonimus westermani, a lung fluke causing human paragonimiasis, secretes a large amount of cysteine proteases, which are involved in nutrient uptake, tissue invasion, and modulation of hos's immune responses. There is, however, limited information about the response of eosinophils to direct stimulation by cysteine proteases (CP) secreted by P. westermani. In the present study, we tested whether degranulation and superoxide production from human eosinophils can be induced by stimulation of the 2 CP (27 kDa and 28 kDa) purified from excretory-secretory products (ESP) of P. westermani newly excysted metacercariae (PwNEM). A large quantity of eosinophil-derived neurotoxin (EDN) was detected in the culture supernatant when human eosinophils isolated from the peripheral blood were incubated with the purified 27 kDa CP. Furthermore, the 27 kDa CP induced superoxide anion production by eosinophils in time- and dose-dependent manners. In contrast, the purified 28 kDa CP did not induce superoxide production and degranulation. These findings suggest that the 27 kDa CP secreted by PwNEM induces superoxide production and degranulation of human eosinophils, which may be involved in eosinophil-mediated tissue inflammatory responses during the larval migration in human paragonimiasis.     

A new species of Paragonimus (Trematoda: Troglotrematidae) from a cat infected with metacercariae from mountain crabs Larnaudia larnaudii

The identification of Paragonimus spp. lung flukes is based on the morphology of both the metacercaria and the adult. A very small Paragonimus sp. metacercaria was found in a freshwater crab caught in Kanchanaburi Province, West Thailand, an area where metacercariae of Paragonimus heterotremus had not been found. The metacercariae cysts were 180-204 microm in diameter, which was smaller than metacercariae of P. heterotremus. The coefficient of difference in body size between this metacercaria and P. heterotremus was 1.69, which was greater than a subspecific difference. Adults recovered from the lungs of a cat fed with the metacercariae were morphologically similar to, but smaller than, P. heterotremus dissected from the lungs of a feline experimental host. The tegumental spines of the worm in this study were singly spaced in arrangement, which is similar to, but larger than, the spines of P. heterotremus. Therefore, Paragonimus pseudoheterotremus is proposed as a new species.     

Metacercariae of Galactosomum lacteum (Jägerskiöld, 1896) Looss, 1899 (Heterophyidae) from marine teleosts in the Gulf of Cagliari (southern Sardinia, Italy)

Galactosomum lacteum (J?gerski?ld, 1896) Looss, 1899 metacercariae, encysted on the optic nerve, on the brain and/or on the muscle and the connective of the pharynx and oesophagus, were found in Spicara maena L., S. flexuosa Rafinesque, 1810, S. smaris L. (Centracanthidae), Gobius cruentatus Gmelin, 1789 (Gobiidae), Symphodus tinca L., S. mediterraneus L. (Labridae), Serranus cabrilla L. (Serranidae), Diplodus sargus L. and D. annularis L. (Sparidae) caught in the Gulf of Cagliari (southern Sardinia, Italy). Excysted specimens were identified by some distinctive morphological features: more or less expanded forebody, depending on whether the specimens were living or fixed; tubular excretory bladder extending to the posterior border of the ovary; two-chambered seminal vesicle; asymmetrical and parenchymatous ventral sucker with lines of spines within its cavity; and unarmed gonotyle. Comparison has been made with the congeneric species metacercaria, G. timondavidi Pearson & Prévot, 1971, also registered in the Mediterranean Sea.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Zygocotyle lunata

High-performance thin-layer chromatography was used to analyze the neutral lipids in the rediae, cercariae, and encysted metacercariae of the paramphistomid trematode Zygocotyle lunata. Visual observations of the chromatograms showed that the most abundant lipid fractions were free sterols and free fatty acids in all larval stages and triacylglycerols in the metacercariae and rediae. The weight of free sterols (x +/- SE) was 120+/-20 ng/cercaria, 56+/-3.8 ng/redia, and 5.9+/-1.5 ng/encysted metacercaria; the weight of triacylglycerols was 13+/-0.88 ng/encysted metacercaria, 6.3+/-0.063 ng/redia, and was not detectable in the cercaria; the weight of free fatty acids was 160+/-17 ng/ cercaria, 76+/-9.1 ng/redia, and 4.2+/-0.46 ng/encysted metacercaria. Oil red O staining of whole larvae showed the presence of neutral lipids in the rediae but not in the cercariae or encysted metacercariae. A dramatic reduction was seen in the quantity of free sterols and free fatty acids in the encysted metacercariae as compared with the cercariae, suggesting that these neutral lipids are used in some way during the transformation from cercaria to metacercaria.     

Studies on host specificity in Paragonimus westermani: I. Ultrastructural characterization of metacercariae and juvenile and adult worms from abnormal and definitive hosts

Comparative ultrastructural studies have been made of newly excysted metacercariae and worms recovered from abnormal (rats) and normal definitive hosts (dogs). Worms from rats survived for prolonged periods without any growth or development in muscles of the host. They appeared to resemble metacercariae in general features, although ultrastructural observations revealed differences in the composition of tegumental granules and the development of the excretory bladder and caeca. Worms from dogs showed well-developed morphology in association with active energy metabolism and biosynthesis.     

Redescription and surface ultrastructure of Pygidiopsis macrostomum (Digenea: Heterophyidae)

Pygidiopsis macrostomum Travassos, 1928, a poorly known species originally described from a single specimen from Rattus norvegicus (Erxleben, 1777) in Brazil, is redescribed on the basis of metacercariae from the mesenteries of naturally infected guppies Poecilia vivipara Bloch and Schneider, 1801 (Poeciliidae), and adults obtained from an experimental infection of hamsters. Pygidiopsis macrostomum is characterized by the absence of oral spines, vitellaria extending forward to ventral sucker, uterus reaching pharyngeal level, X-shaped excretory vesicle, and an oral sucker/acetabulum ratio of 1:0.8. The surface ultrastructure shows that the tegument of the metacercaria does not strongly differ from that of adults. The brush-shaped spines of P. macrostomum are similar to those reported for Pygidiopsis summa and Pygidiopsis ardeae, but no differences in spine shape were observed throughout the body.     

Occurrence of a diploid type and a new first intermediate host of a human lung fluke, Paragonimus westermani, in Korea

The biology, chromosome number, and karyotype of a lung fluke, Paragonimus westermani (Kerbert, 1878) collected in Haenam, Haenam-gun Chollanam-do, Korea were analyzed. We compared the size of metacercariae from Haenam with those taken from a crayfish collected at Youngam, Youngam-gun, Chollanam-do, Korea. The mean length of P. westermani metacercariae from Haenam was 300.3 microm and that from Youngam was 362.0 microm. Adult worms were recovered from the lungs of experimentally infected dogs. The mean egg sizes obtained from adult flukes were 72.1 x 46.8 microm from Haenam and 93.5 x 54.2 microm from Youngam. Semisulcospira tegulata collected in the Youngam area were found to be infected with cercariae of P. westermani, one of the snail-borne human lung fluke trematodes in Korea. Of 4218 snails studied, 5 (0.12%) harbored P. westrermani larvae. This is the first report of S. tegulata serving as the initial intermediate host of P. westermani. The chromosome numbers of P. westermani from Haenam and Youngam were 2n = 22 and 3n = 33. The diploid type of P. westermani has not been previously reported in Korea.     

A new cercaria and metacercaria of Acanthoparyphium (Echinostomatidae) found in an intertidal snail Zeacumantus subcarinatus (Batillaridae) from New Zealand

A new 23-collar-spined cercaria and metacercaria are described from intertidal molluscs of the coast of New Zealand. The new cercaria found emerging from the mud snails Zeacumantus subcarinatus (Sowerby) (Prosobranchia: Batillariidae) is characterized mainly by the number and arrangement of the cephalic glands, the size of the suckers, and the size and number of the collar spines. The cercaria encysts in the cockle Austrovenus stutchburyi (Wood) (Bivalvia: Veneridae) which lives in sympatry with the first intermediate host throughout New Zealand. Laboratory infections of cockles by cercariae from naturally infected snails resulted in metacercariae identical to those found in naturally infected cockles. The main features of the cercaria and metacercaria are the presence of a reniform collar with 23 spines, two pairs of small cephalic glands at the oral sucker level, another two pairs of much longer ones posterior to the pharynx, and the excretory vesicle Y-shaped with the main collecting canals extending to the anterior level of the ventral sucker. Each of the main collecting canals had 10-11 pairs of bilateral diverticula between the anterior edge of the ventral sucker and anterior body end. A brief discussion of its possible life cycle and ecology is also provided.