Cloning of a cysteine proteinase cDNA of adult Paragonimus westermani by polymerase chain reaction.

Cysteine proteinase cDNA fragment from adult mammalian trematode, Paragonimus westermani was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 804 bp. The deduced amino acid sequence consists of 268 amino acids. Sequence analysis and alignment showed significant sequence similarity to other eukaryotic cysteine proteinases and conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. The cysteine proteinase cDNA fragment was also subcloned in the expression vector pET and expressed as a C-terminal His-tag fusion protein in Escherichia coli.

Cloning of a cysteine proteinase gene from Acanthamoeba culbertsoni.

Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more active in pathogenic strains of amoeba whereas serine proteinases are found in both pathogenic and nonpathogenic strains. Cysteine proteinases thus play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culbertsoni by performing reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 1359 bp. The deduced amino acid sequence has the sequence homology with the cysteine proteinase genes of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathepsin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respectively. Sequence analysis and alignment showed significant similarity to other eukaryotic cysteine proteinases, including the conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. A 1.5 kbp mRNA was detected on Northern blot analysis using full-length cysteine proteinase cDNA as a probe. The A. culbertsoni cysteine proteinase gene (AcCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/threonine-rich C-terminal extension. Therefore, it has cathepsin L-like characteristics. Phylogenetic analysis based on the amino acid sequences of cysteine proteinase indicated that AcCP2 was closely related with papaya, while it was remotely related with those of Schistosoma.

Effect of iron on the virulence of Trichomonas vaginalis.

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T. vaginalis is reduced.

Genotyping of Giardia lamblia isolates from humans in China and Korea using ribosomal DNA Sequences.

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsimony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.

Diagnosis of trichomoniasis by polymerase chain reaction.

The clinical usefulness of polymerase chain reaction (PCR) for the diagnosis of trichomoniasis was evaluated in comparison with other conventional tests. PCR was used for specific detection of Trichomonas vaginalis by primers based on the repetitive sequence cloned from T. vaginalis (TV-E650). Between June 1996 and August 1997, 426 patients visited the department of obstetrics and gynecology, Hanyang University Kuri Hospital and were examined for trichomoniasis using wet mount examination, Papanicolaou (Pap) smear, culture and PCR. One hundred and seventy-seven patients (group A) visited with the symptoms of vaginal discharge and 249 patients (group B) visited for regular cervical Pap smear with no vaginal symptoms. From group A (n = 177), 3 infections (2.0%) were detected by wet mount, 6 infections (3.3%) by Pap smear and culture, and 17 infections (10.4%) by PCR. From group B (n = 249), 4 patients (1.6%) were found to have T. vaginalis by culture and 6 infections (2.4%) were detected by PCR. Therefore, in both groups, PCR for T. vaginalis showed a higher detection rate compared with conventional wet mount, Pap smear or culture. The detection by PCR was specific for T. vaginalis since no amplification was detected with DNAs from other protozoa and Candida albicans. The sensitivity and specificity of PCR were 100%. This method could detect T. vaginalis in vaginal discharge at a concentration as low as 1 cell per PCR mixture. These results indicate that PCR could be used as a specific and sensitive diagnostic tool for human trichomoniasis.

Synthesis of praziquantel derivatives and their in vitro activity against adult Clonorchis sinensis.

Several praziquantel derivatives have been prepared by the acylation of compound 5, and examined on their biological activity in vitro a against adult Clonorchis sinensis collected from rabbits infected with metacercariae which was isolated from Pseudorasbora parva, a second intermediate host, captured in Nakdong river in Korea.

Phylogenetic relationships between the six superoxide dismutase proteins (FeSOD) of Trichomonas vaginalis and FeSOD6 genetic diversity.

The parasitic protozoan Trichomonas vaginalis is known to contain several types of Fe-containing superoxide dismutase proteins (FeSOD). Using three different methods of phylogenetic analysis, maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods, we examined the phylogenetic relationships among the six FeSOD (FeSOD1-FeSOD6) based on their amino acid sequences. All the analyses consistently suggested that the six proteins formed a monophyletic group implying that they probably be originated from an ancestral protein form through repeated duplication events. Although MP tree was totally unresolved, the NJ and ML trees revealed that FeSOD6 placed the most basal position and thus emerged earlier than the other five gene types during the evolution of T. vaginalis. Phylogenetic relationships among the five remaining proteins were (FeSOD2, FeSOD3), (FeSOD4, (FeSOD1, FeSOD5)) although weakly supported in terms of bootstrapping values. In addition to this, we newly designed two PCR primer specifically amplifying full-length FeSOD6 gene and examined its genetic diversity among 12 T. vaginalis isolates from five countries and three continents. They had the same nucleotide sequences except those of three Korean isolates which showed one to three different nucleotides.

Molecular identification of Paragonimus ohirai and P. westermani from Anhui Province, China.

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) region were determined from seven adults of species Paragonimus collected from Jinde and Xiuning Counties, Anhui Province, China. Among these, the nucleotide sequence obtained from one Paragonimus adult (Jinde County) was identical to the ITS2 sequence of P. ohirai previously reported. In order to confirm the result, partial regions of mitochondrial cytochrome C oxidase I (COI) and NADH dehydrogenase 1 (ND1) from the putative P. ohirai sample were further sequenced. They showed a high level of similarity with those of P. ohirai, COI (99.7%) and ND1 (99.5%), supporting the result obtained from the ITS2. In addition to this, we designed P. ohirai- and P. westermani-specific primers (BDW and BD2OH) from ITS2 to identify P. westermani and P. ohirai easily and rapidly. After testing utility of the primers, they were applied to identify seven unidentified Paragonimus samples collected from Jinde and Xiuning Counties, China. All the examined samples showed P. westermani band pattern, and it was reconfirmed by sequencing their ITS2 regions that they are P. westermani. This result indicates that the two newly designed specific primers could be quite helpful for easily identifying P. westermani and P. ohirai, that most of Paragonimus in Jinde and Xiuning Counties consist of P. westermani, and that P. ohirai exists in Jinde County with minority.

Trichomonas vaginalis promotes apoptosis of human neutrophils by activating caspase-3 and reducing Mcl-1 expression.

Neutrophils are the predominant inflammatory cells found in the vaginal discharge of patients with Trichomonas vaginalis infection. However, it is not known whether neutrophil apoptosis is induced by live T. vaginalis. Therefore, we examined whether T. vaginalis can influence neutrophil apoptosis, and also whether caspase-3 and the Bcl-2 family members are involved in the apoptosis. Thus, human neutrophils were incubated with live T. vaginalis and neutrophil apoptosis was evaluated by Giemsa, annexin V-PI, and DiOC6 stainings. The neutrophil apoptosis was significantly higher in those incubated with T. vaginalis than in the control group. When trichomonads were pre-treated with mAb to AP65 (adhesin protein), or when trophozoites were separated from neutrophils using a Transwell chamber, neutrophil apoptosis was significantly reduced. The activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis but was markedly enhanced during T. vaginalis-induced apoptosis. Moreover, the inhibition of caspase-3 effectively reduced T. vaginalis-induced apoptosis. Trichomonad-induced apoptosis was also associated with reduced expression of the neutrophil anti-apoptotic protein, Mcl-1. These results indicate that T. vaginalis alters Mcl-1 expression and caspase-3 activation, thereby inducing apoptosis of human neutrophils.

Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos.

Faecal examinations for helminth eggs were performed on 1869 people from two riverside localities, Vientiane Municipality and Saravane Province, along the Mekong River, Laos. To obtain adult flukes, 42 people positive for small trematode eggs (Opisthorchis viverrini, heterophyid, or lecithodendriid eggs) were treated with a 20-30 mg kg(-1) single dose of praziquantel and purged. Diarrhoeic stools were then collected from 36 people (18 in each area) and searched for helminth parasites using stereomicroscopes. Faecal examinations revealed positive rates for small trematode eggs of 53.3% and 70.8% (average 65.2%) in Vientiane and Saravane Province, respectively. Infections with O. viverrini and six species of intestinal flukes were found, namely, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus caninus, Prosthodendrium molenkampi, and Phaneropsolus bonnei. The total number of flukes collected and the proportion of fluke species recovered were markedly different in the two localities; in Vientiane, 1041 O. viverrini (57.8 per person) and 615 others (34.2 per person), whereas in Saravane, 395 O. viverrini (21.9 per person) and 155207 others (8622.6 per person). Five people from Saravane harboured no O. viverrini but numerous heterophyid and/or lecithodendriid flukes. The results indicate that O. viverrini and several species of heterophyid and lecithodendriid flukes are endemic in these two riverside localities, and suggest that the intensity of infection and the relative proportion of fluke species vary by locality along the Mekong River basin.

Cestode infections in Korea.

Epidemiological surveys concerning cestodes of medical importance in Korea have been sporadically reported by many workers. Among various parasitic infections, cestode infections occur less frequently than other helminthiases, but they often cause serious clinical complications. Cestodes reported so far in Korea belong to the Order Pseudophyllidea or Cyclophyllidea (Subclass Cestoda), i.e., Diphyllobothrium latum, D. yonagoense, Spirometra erinacei, sparganum, Hymenolepis diminuta, H. nana, Mesocestoides lineatus, Taenia saginata, T. solium, metacestode of T. solium (cysticercus), and Echinococcus granulosus. The present paper is a brief review of the literature concerning these cestode infections in Korea. Taeniases by T. saginata or T. solium are prevalent cestode infections in Korea, and cysticercosis by T. solium often reveals serious clinical manifestations. H. nana infection is relatively rare, showing 0.22% egg positive rate in a nationwide survey in 1986. Sparganosis by the plerocercoid larva of Spirometra is not an uncommon cestode infection in man, and 16 kinds of animal hosts including the snakes and frogs have been reported. D. latum infection has been an uncommon intestinal parasitosis, but it tends to increase nowadays. Most of human hydatidosis cases have been recognized as imported ones from outside Korea, but one case was proven indigenous. Other 4 kinds of cestodiases are extremely rare in Korea and only a few cases have been recorded.

Remarks on the diagnosis of Clonorchis sinensis infection.

A number of techniques have been developed for the diagnosis of Clonorchis sinensis infection. Although the most reliable technique for this infection are the various stool examinations in the laboratory, practical problems such as difficulties of stool collection and mass examinations need more applicable methods for epidemiological studies. During past decades some serologic and immunologic methods such as intradermal skin test, gel-diffusion technique, complement fixation test, haemagglutination test, fluorescent antibody test and enzyme-linked immunosorbent assay (ELISA) have been introduced for the diagnosis of clonorchiasis in Korea, but satisfactory results have not been obtained especially in field work because of the lack of available purified antigen. From this viewpoint it is essential to undertake careful isolation and purification of specific antigens. Recently ELISA has been well established as a micro-system for mass survey. In spite of the complexity of its procedures in contrast to the skin test the results obtained explain its importance. In this paper available serologic and immunologic methods for the diagnosis of clonorchiasis used at the Institute of Tropical Medicine, Yonsei University, Seoul, are reviewed, and the results from skin test and ELISA are compared and discussed.

[Resistance to Naegleria fowleri infection passively acquired from immunized splenocyte, serum or milk].

A pathogenic free-living amoeba, Naegleria fowleri, causes primary amoebic meningoencephalitis to human and experimental animals. This infection is rare, but the mortality is very high. Nowadays, drug treatment or active immunization of human or mice are being tried with partial effectiveness. This study shows passive immunization effect by transfer of immunized spleen cells, serum, or milk from immunized mother in mouse experimental model. Young BALB/c mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri, and spleen cells and sera were collected for injection to recipient mice. There were seven transfer groups, i.e., immunized mouse serum, spleen cells, serum and spleen cells, normal mouse serum, spleen cells, serum and spleen cells, and control group. Three days later, BALB/c mice were inoculated with 1 x 10(4) trophozoites of N. fowleri intranasally. After infection, decreased mortality and prolonged survival time of mice were noted in immunized groups compared with non-immunized control group. The groups injected with immunized spleen cells or normal serum showed lower mortality than that of controls but showed no changes of serum IgG level. The groups injected with immunized serum or normal spleen cells showed increased serum IgG level after immunization but hundred percent mortality was observed. Mother mice were immunized intraperitoneally with 2-3 X 10(6) trophozoites of N. fowleri at the end of pregnancy and weaning period. Soon after the delivery, litters born of non-immunized mother were matched with immunized mother for feeding immune milk. After three weeks, the litters were infected with 1 X 10(4) trophozoites of N. fowleri or sacrificed for serum collection to measure the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of interferon-gamma and interleukin-4 by splenocytes in mice infected with Paragonimus westermani.

The TH cytokine responses of spleen cells stimulated with Con A from mice infected with Paragonimus westermani were examined. The spleen cell culture supernatants were assayed for TH1-specific IFN-gamma and TH2-specific IL-4. Cytokine responses for IL-4 peaked at three days (410 +/- 60.9 pg/ml), persisted at a high level until the second week (343 +/- 59.0 pg/ml), and then decreased slowly four and six weeks after infection. IFN-gamma production by splenocytes only increased during the first week (151 +/- 32.3 pg/ml) and declined abruptly after the second week of infection. IFN-gamma production by splenocytes of infected mice was not observed during the sixth week of infection. In addition, serum IL-4 and IFN-gamma were measured. Serum IL-4 was not detected in substantial quantity until four to six weeks after infection. The time course of serum IL-4 was not correlated with that of IL-4 production by splenocytes. Serum IFN-gamma was undetectable during the entire course of infection. These results suggest that TH2 cytokine responses, rather than TH1, predominate in mice infected with P. westermani.

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.

Changes of IgE production, splenic helper and suppressor T lymphocytes in mice infected with Paragonimus westermani.

Effects of Paragonims westermani infection were observed in mice on the change of serum IgE level, the number of peripheral eosinophils and the distribution of Thy 1.2+ (CD3), L3T4+ (CD4), and Lyt-2+ (CD8) splenic T lymphocytes without mitogen stimulation. BALB/c mice were infected with 20 metacercariae of P. westermani each. Total serum IgE increased at 3 weeks after the infection and reached a peak on week 4 and maintained high levels of IgE until the 23rd week. Peripheral eosinophil numbers increased at the second week and attained peak level on week 9. The frequency of L3T4+ (CD4) and Lyt-2+ (CD8) T lymphocytes decreased slightly until 4 weeks after the infection, but not significantly. Absolute number of L3T4+ and Lyt-2+ T lymphocytes, and the ratio of L3T4/Lyt-2 were not markedly changed over the period of observation. The frequency of Thy 1.2+ (CD3) T lymphocytes in the infected group slightly decreased until 4 weeks after the infection and showed significant reductions at the 2nd and 4th week of the infection (p < 0.05).

[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.

[Cytotoxicity of lymphokine activated peritoneal macrophages against Trichomonas vaginalis].

Trichomonas vaginalis is a parasitic flagellate in the urogenital tract of human. Innate cytotoxicity of macrophages against T. vaginalis has been recognized, but any report on the cytotoxicity of lymphokine-activated macrophages to T. vaginalis is not yet available. The present study aimed to elucidate the lymphokine-activated cell mediated cytotoxic effect against T. vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured by counting the release of 3H-thymidine from prelabeled protozoa, and tested in U-bottom microtiter plates. Nitrite concentration in culture supernatants was measured by standard Griess reaction. The results obtained are as follows: 1. The cytotoxicity of macrophages was increased by addition of rIL-2 or rIFN-gamma. 2. Cytotoxicity of macrophages was reduced by addition of rIL-4 to rGM-CSF, rIL-2 or rIFN-gamma. 3. Crude lymphokine mixed with anti-IL-2 decreased the cytotoxicity of macrophages. 4. In case of macrophages cultured with rIFN-gamma or rIL-4, the concentration of nitrite was related with cytotoxicity of macrophages against T. vaginalis, but the cytotoxicity of macrophages cultured with rIL-2 and rIFN-gamma was decreased in spite of its high production of nitrite. From the results obtained, it is assumed that rIL-2 and rIFN-gamma enhance the cytotoxicity of macrophages while rIL-4 inhibits the cytotoxicity against T. vaginalis, and that the production of nitrite does not relate with the cytotoxicity of macrophages, but nitric oxide may play a role as an inhibitory factor on the proliferation of T. vaginalis.

[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.