Excretory-secretory product of newly excysted metacercariae of Paragonimus westermani directly induces eosinophil apoptosis

Eosinophils are important effector cells in host defense against parasites. Excretory-secretory product (ESP) produced by helminthic worms plays important roles in the uptake of nutrients, migration in the host tissue, and in immune modulation. However, little is known about the ability of the ESP to directly trigger eosinophil apoptosis. This study investigated whether the ESP of newly excysted metacercariae of Paragonimus westermani could induce apoptosis in human eosinophils. Apoptosis was assayed by staining the cells with FITC-annexin V, and the cells were analyzed by flow cytometry. It was found that the ESP of newly excysted metacercariae of P. westermani induced a direct time- and concentration-dependent increase in the rate of constitutive apoptosis in mature human eosinophils. Eosinophil apoptosis was first apparent 3 hr after treatment with the ESP and continued to increase after 6 hr of incubation with respect to the cells cultured in the absence of the ESP. While only 2.8% of the eosinophils incubated in the medium for 3 hr were apoptotic, 7.6%, 10.9% and 22.6% of the eosinophils treated with 10, 30 and 100 micrograms/ml ESP were apoptotic, respectively. This result suggests that the ESP of newly excysted metacercariae of P. westermani directly induce eosinophil apoptosis, which may be important for the survival of the parasites and the reduction of eosinophilic inflammation in vivo.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Early cysteine protease activity in excretory bladder triggers metacercaria excystment of Paragonimus westermani

The cysteine proteases of Paragonimus westermani metacercaria are known to initiate metacercaria excystment. However, their secretory sites, such as the intestine, tegument, and excretory bladder are not well defined. In this study, the metacercariae were labeled with bromodeoxyuridine (BrdU) to distinguish the initial activation sites. BrdU was labeled mainly at the excretory bladder and the excretory granules of the metacercariae and the newly excysted juvenile worms. This result shows that early 'defecation' of the proteases from the excretory bladder may accelerate the excystment of P. westermani metacercariae.     

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.     

Effect of cobalt-60 irradiation on the infectivity of Paragonimus westermani metacercariae.

A study was made to observe the effect of cobalt-60 irradiation on the viability of Paragonimus westermani metacercariae in Sinopotamon chekiangense crabs. The crabs were collected in mountain regions of the Zhejiang Province of China in which paragonimiasis is endemic. Adult cats and albino mice were infected with metacercariae irradiated at different doses. Dissection of the host animals was conducted 90 or 30 days, respectively, after infection for recovery of lung flukes. Anti-metacercariae antibody in infected mice was measured by enzyme-linked immunosorbent assay (ELISA). Results showed that metacercariae were unable to grow into adult worms in cats after exposure to gamma irradiation at a dose of 0.10 kGray. However, a small number of metacercariae exposed to a dose of 2.0 kGray excysted and survived in 1 mouse for 30 days. No worm was recovered from mice when the metacercariae were irradiated at a dose of 2.5 kGray. Seropositive results by ELISA were obtained when the mice were infected with metacercariae irradiated at doses ranging from 2.0 to 3.5 kGray.     

Characterization and localization of Paragonimus westermani antigen stimulating antibody formation in both the infected cat and rat

Applicability of the adult Paragonimus westermani antigen for detection of anti-immature P. westermani antibodies in experimentally infected rats, a paratenic host of this lung fluke, was examined. The serum antibodies of the cats and rats infected with P. westermani metacercariae were detected by enzyme-linked immunosorbent assay (ELISA) with the adult-fluke antigen. The ELISA titers of serum samples of the rats infected with only immature flukes were as high as those of the cats infected with adult flukes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and the immunoblotting technique showed that a major protein band of 27,000 daltons was recognized in the sera of the infected cats and rats. Immunoperoxidase staining applied on the sectioned flukes provided evidence showing that the antigenic substance was located on the surface of the gut epithelium and in the luminal contents in both adult and immature flukes. The adult-fluke antigen containing the 27,000-dalton substance is applicable as a standard antigen for diagnosis of paragonimiasis westermani in not only definitive hosts but also in paratenic hosts.     

A neutral thiol protease secreted from newly excysted metacercariae of trematode parasite Paragonimus westermani: purification and characterization

1. A neutral thiol protease was purified from the culture filtrate of newly excysted metacercariae of Paragonimus westermani to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a monomeric form with mol. wt 22,000. 2. It expressed activity on t-butyloxycarbonyl-valyl-leucyl-lysyl-4-methyl-coumaryl-7-amide in the presence of cysteine at an optimal pH of 7.5, and also the activity was significantly affected by thiol protease inhibitors, indicating that the enzyme belongs to a neutral thiol protease family. 3. The enzyme hydrolyzed protein substrates, azocoll, casein and fluorescein isothiocyanate-labeled collagen, and showed low specificity toward hemoglobin, but no activity with elastin Congo Red and bovine serum albumin. 4. Catalytic property on fluorogenic substrates demonstrated that the enzyme cleaved preferentially the carboxylic side of the basic residue in N-substituted peptides.     

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.     

Excretory-secretory product of Paragonimus westermani newly excysted metacercariae inhibits superoxide production of granulocytes stimulated with IgG

It is well known that the cysteine proteases in excretory-secretory product (ESP) of Paragonimus westermani newly excysted metacercariae (PwNEM) are capable of degrading IgG in vitro. Recent evidence suggests that the IgG-coated surface, such as found on parasites, is one of the most effective physiologic stimuli for granulocyte activation. Therefore, this study was designed to investigate the effect of excretory-secretory product (ESP) of PwNEM on superoxide production of granulocytes stimulated with IgG. The 96-well plates were coated with human IgG (0, 10, 30, 100 micrograms/ml) in the absence or presence of ESP. When granulocytes were incubated in the wells coated with human IgG in the presence of ESP, the level of superoxide production of granulocytes was reduced to about 90% when compared to the cells incubated in the wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production of granulocytes was concentration-dependent. These results suggest that ESP secreted by PwNEM may be important in the control of effector functions of granulocytes stimulated with IgG in human paragonimiasis.     

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.     

The cell content of peritoneal exudates following the injection of neutral thiol protease into guinea pigs.

The cell content of the peritoneal exudate was examined 4 days after an intraperitoneal injection of glycogen in uninfected and Paragonimus westermani-infected guinea pigs. In uninfected animals a reduction in macrophage count and an accumulation of granulocytes in the exudate were observed at 3 and 6 hr after an intraperitoneal injection of purified neutral thiol protease from P. westermani metacercariae. No such effect occurred after the enzyme was injected into infected animals. At 9 hr after enzyme injection, vacuoles were found in the cytoplasm of macrophages in uninfected animals.     

Serum IgE levels in rats infected with Paragonimus westermani]

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.     

Human paragonimiasis in Viet Nam: epidemiological survey and identification of the responsible species by DNA sequencing of eggs in patients' sputum

Parasitological and sero-epidemiological surveys for human paragonimiasis were conducted in three provinces of Viet Nam. A total of 590 participants from two known endemic areas of human paragonimiasis (Sinho district of Laichau province and Lucyen district of Yenbai province) and from Dakrong district of Quangtri province where we recently found crab hosts heavily infected with Paragonimus westermani metacercariae. By multiple dot-ELISA screening, 28 (12.7%) out of 220 participants in Sinho district of Laichau province and 4 (3.3%) out of 120 participants in Lucyen district of Yenbai province were proven to be antibody-positive against the Paragonimus antigen. None of the 250 sera of the residents in Dakrong, Quangtri province, gave sero-positivity. Among a total of 32 sero-positive patients Paragonimus eggs were found in 6 cases. ITS2 sequences were successfully determined from a single Paragonimus egg from each patient. The results of homology search by BLAST and alignment clearly confirmed that Paragonimus eggs collected from 6 patients were all of Paragonimus heterotremus. The pathogenicity of P. westermani for human paragonimiasis in Viet Nam is still questionable and needs to be explored in the future.     

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.     

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.     

Tegumental ultrastructures of Paragonimus iloktsuenensis according to the developmental stages

A scanning electron microscopic study was performed to observe the tegumental ultrastructures of Paragonimus iloktsuenensis according to its developmental stages. The metacercariae were obtained from the liver of the brackish water crab, Sesarma dehaani. Juvenile and adult P. iloktsuenensis were recovered from the experimental rats on 2, 4 and 8 weeks after infection. The findings were summarized as follows: 1. The excysted metacercariae were characteristically gourd-shape, with their whole body surface beset with numerous spade-shape spines. The large, type II sensory papillae (non-ciliated round swellings) were arranged along the rim of the oral and ventral suckers, 11-12 and 6-8 in numbers respectively. 2. Two-week old juvenile worms, recovered chiefly from the liver of the experimental rats, were slender in body shape, with their ventral sucker near the anterior one-third level. The distribution of tegumental spines was less dense than in the excysted metacercariae. The spines were with 1-2 pointed tips and 3-4 longitudinal splits. Numerous ciliated knob-like, type I papillae were observed in both sides of the oral sucker, and 6 large, type II papillae were arranged along the rim of the ventral sucker. 3. Four-week old worms, recovered from the thoracic cavity and/or lung parenchyme of the experimental rats, were thicker than wide in body configuration, and their ventral sucker was located near the anterior one-fourth level. The tegumental spines at ventral surface were grouped, each group with 3-5 aggregated ones. The type I and type II papillae (small-sized) were distributed chiefly around the rim of two suckers. 4. Adult (eight-week old) worms, recovered from the capsules in the lung parenchyme, were very stout, and covered densely with bearfoot-like spines. At dorsal surface, cobblestone-like cytoplasmic processes were well-developed, with many tegumental spines embedded in them. It was observed in this study that the tegument of P. iloktsuenesis continued to change and differentiate as the worms grew to be adults.     

Identities of two Paragonimus species from Sri Lanka inferred from molecular sequences

Metacercariae of Paragonimus spp. were obtained from field-collected freshwater crabs in Sri Lanka. Genomic DNA was extracted from single metacercariae. Two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified using the polymerase chain reaction. Two differing sequences were obtained for each of these gene regions. Phylogenetic analyses placed the type 1 sequences as sister to a clade containing P. westermani and P. siamensis whereas the type 2 sequences were close to published sequences of P. siamensis from Thailand. The possible taxonomic status of these two types are discussed. This is the first report of molecular data about Paragonimus from Sri Lanka.     

Co-existence of Paragonimus harinasutai and Paragonimus bangkokensis metacercariae in fresh water crab hosts in central Viet Nam with special emphasis on their close phylogenetic relationship

During our epidemiological surveys for Paragonimus species in central Viet Nam, we found four morphologically different Paragonimus metacercariae in mountainous crabs. They were identified as metacercariae of Paragonimus westermani, P. bangkokensis, P. proliferus, and P. harinasutai in the order of their prevalence in crab hosts. This is the first discovery of P. harinasutai in Viet Nam, co-inhabiting with P. bangkokensis and other species. Metacercariae of P. harinasutai were given orally to a cat to obtain adult worms. Then, ITS2 and CO1 sequences of metacercariae and adults of P. harinasutai, and metacercariae of P. bangkokensis collected from the same place were determined for analyses of phylogenetic relationships to other P. harinasutai and P. bangkokensis populations as well as related species. The results of molecular analyses showed that P. harinasutai from Quang Binh province of central Viet Nam was almost completely identical with those from Vientiane, Lao PDR; P. bangkokensis from Quang Binh, Viet Nam was also almost completely identical with those from Lao PDR and from Quang Ninh province, Viet Nam. Except for one P. harinasutai isolate from China, all populations of P. harinasutai and P. bangkokensis from Thailand, Lao and Viet Nam make a single clade in both ITS2 and CO1 trees. In ITS2 sequences, AT deletion and ATC insertion were observed in some isolates of both species, indicating recent gene flow between P. harinasutai and P. bangkokensis. Moreover, because of their extremely high genetic similarities and their co-inhabitation in the same crab hosts found in Thailand, Lao PDR and Viet Nam, they should be considered as the sister species at the early stage of divergence. In addition, P. microrchis previously described from Yunnan, China should be placed as the synonym of P. harinasutai, because of their morphological and molecular similarities.