Production and Deformation of Clonorchis sinensis Eggs during In Vitro Maintenance

Clonorchis sinensis is a carcinogenic human liver fluke. The present study monitored eggs produced by long-term maintained adult worms of C. sinensis to confirm their egg productivity in vitro. The worms from infected rabbits were incubated in vitro in 1× Locke’s solution and broth media (RPMI-1640, DMEM and IMDM). Numbers of expelled eggs were counted sequentially and their morphological changes were monitored by microscopy after 1, 30, 60, and 90 days of cultivation. On the 1–3 days of cultivation, the eggs counted maximum 4,756±202 eggs/worm/day in IMDM medium. The number of eggs gradually decreased less than 1,000 at 7–14 days and below 100 at 21days but continued to pass eggs after 56 days in all media. Length of the eggs were reduced about 1 µm at 30 days, and the length/width ratio was maintained around 1.8 at 30 days but decreased to 1.7 at 60 days and 1.5 at 90 days. Faust-Meleney index (FMI) decreased as the cultivation duration increased and lowest FMI (5662.9±974.7) observed in IMDM media at day 90 (P = 0.001). Microscopic findings of the eggs recognized the miracidium in most of eggs at 60 days but not in those at 90 days. Instead, the eggs contained dark granules or vacuoles in the deformed shell at 90 days. Scanning electron microscopy revealed partial loss of wrinkles on the deformed egg surface and prominent abopercular knob. Eggs viability decreased as the cultivation progressed and showed significant positive correlation with FMI and length/width ratio. In conclusion, the cultivated worms pass only the eggs which are preformed in their uterus before cultivation. One gravid C. sinensis contains about 37,000 eggs in its uterus and produces about 4,000 eggs every day. The deformed eggs with FMI less than 7,000 and length/width ratio lower than 1.7 are non-viable.     

Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.     

The biochemical and immunological characterization of two serpins from Clonorchis sinensis

Serpins (serine proteinase inhibitors) are evidenced to regulate numerous biological processes such as immunoregulation in parasitic helminths. The functions of serpins from Clonorchis sinensis remain unclear to date. In this study, two serpin genes, respectively denominated as CsproSERPIN and CsSERPIN2, had been selected from metacercaria cDNA library of C. sinensis. The biochemical activities of both recombinant proteins (rCsproSERPIN and rCsSERPIN2) were analyzed by assays of inhibition on some serine or cysteine proteases, the results showed that rCsproSERPIN significantly inhibited trypsin, chymotrypsin and thrombin, while rCsSERPIN2 inhibited only chymotrypsin. Moreover, cytokine and antibody measurements indicated that rats subcutaneously immunized with rCsproSERPIN and rCsSERPIN2 respectively developed a strong IFN-γ production and IgG2a levers of sera were higher than IgG1. Besides, immunoblot assays revealed that the rCsproSERPIN and rCsSERPIN2 could be recognized by the sera of rats infected with C. sinensis and the sera of rabbits immunized by excretory/secretory products. Furthermore, immunofluorescence assays illuminated the two were similarly localized in the reproductive organs such as vitelline glands, testis and eggs in adult stage. In short, all the results collectively indicated that CsproSERPIN and CsSERPIN2 might play important role in the parasite development by preventing the parasite from digestion by exogenous serine proteases, as well as CsproSERPIN and CsSERPIN2 probably involved in immunoregulation of host by inducing Th1-biased type cytokines in rats.     

Expression pattern and substrate specificity of Clonorchis sinensis tyrosinases

Tyrosinase (TYR) is a copper-containing glycoenzyme that mediates hydroxylation of tyrosine into dihydroxyphenylalanine and oxidation of dihydroxyphenylalanine into dihydroxyphenylalanine quinone. TYRs play pivotal roles in eggshell sclerotisation of trematode parasites, while their comprehensive biochemical properties remain elusive. We characterised genes encoding four TYRs (CsTYR1–4) of Clonorchis sinensis, a causative agent of human hepatobiliary disease. These genes shared tightly conserved amino acid residues, two copper binding catalytic motifs and a cysteine-rich epidermal growth factor-like domain. The native and recombinant CsTYRs showed high reactivity against diphenol compounds, especially those with hydroxyl groups in ortho-positions (catechol and l-dihydroxyphenylalanine), but showed minimal activity toward monophenol compounds. Diphenolase activity was enhanced by increased pH of the reaction buffer from 5.0 to 7.0. The temporal induction of CsTYR expression coordinated with the sexual maturation of the worm; enzyme activity was mainly in the vitelline glands and intrauterine immature eggs proximal to the ovary. The primary structures and functional domains of CsTYRs showed significant similarities to those of the vertebrate orthologs, whereas the amino acids shared with the nematode and insect proteins were largely restricted in the bicopper active center. Unlike highly diverged TYR homologs in vertebrates, multiple paralogs have not yet evolved into the separate lineages in trematode genomes, suggesting that duplication of TYR genes might relate to increased genic dosage/redundancy in trematodes. In vitro treatment of copper chelator, diethyldithiocarbamic acid, inhibited generation of phenotypically normal egg. TYR proteins are essential for C. sinensis reproduction, thus might be targeted for therapeutic and vaccine strategies against clonorchiasis, which is prevalent in several Asian countries and is one of the most important predisposing factors for human cholangiocarcinoma. The close phylogenetic relationships between trematode and vertebrate homologs also provide a molecular clue to understand the multifaceted evolutionary pathway of TYR homologs across animal taxa.     

Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum

The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation.     

Nematospiroides dubius in mice selected for liability to infection: Modification of parasite biology through host selection

Brindley P. J. and Dobson C. 1982. Nematospiroides dubius in mice selected for liability to infection: modification of parasite biology through host selection. International Journal for Parasitology12: 573–578. Mice selected as liable (L) and refractory (R) over ten generations voided significantly more and less Nematospiroides dubius eggs compared with randomly mated (Rd) mice after primary infections with 100 larvae. There was little difference between the number of parasite eggs voided g^?1 faeces (epg) by individual mice on day 14 compared with day 15 after infection.However there was a significant diurnal variation in the egg values for individual mice but the mean differences observed between the R, Rd and L mice were maintained over a 24 h period. There was a strong correlation between both the total number and the number of female worms, surviving 21 days after infection, and the mean epg 14 and 15 days after infection. Female N. dubius produced more eggs in L mice and fewer eggs in R mice compared with worms in Rd mice. Similarly, worms grew longer in L mice and were shorter in R mice compared with parasites in Rd mice.     

Studies on metabolism of lung fluke genus Paragonimus. VII. Action of bithionol on glycolytic and oxidative metabolism of adult worms

The present study was carried out in order to obtain information on the mechanism of action of bithionol on Paragonimus westermani (Kerbert 1878). Bithionol stimulated lactic acid production of intact adult worms above the level of the control worms, while it inhibited oxygen consumption of intact adult worms in vitro. Bithionol treatment of adult worms in vivo decreased glycolytic and oxidative metabolism of homogenates of uterine eggs and adult worms.Bithionol inhibited lactic acid formation except when fructose 1, 6-diphosphate (FDP) was used as a substrate in the homogenates of adult worms, and it also inhibited oxygen consumption of homogenates of eggs and adult worms. Bithionol inhibited reduction of methylene blue when succinate was used as a substrate. Bithionol inhibited oxidation of reduced cytochrome c in the 1000g supernatant of homogenates of adult worms. Bithionol inhibited activity of the succinate oxidation in homogenates of adult worms.     

Correlation between discharged worms and fecal egg counts in human clonorchiasis

Background

Stool examination by counting eggs per gram of feces (EPGs) is the best method to estimate worm burden of Clonorchis sinensis in infected humans. The present study investigated a correlation between EPGs and worm burden in human clonorchiasis.

Methods and Findings

A total of 60 residents, 50 egg-positive and 10 egg-negative, in Sancheong-gun, Korea, participated in this worm collection trial in 2006–2009. They were diagnosed by egg positivity in feces using the Kato-Katz method. After administration of praziquantel, they were purged with cathartics on the next day, and then discharged adult worms were collected from their feces. Their EPGs ranged from 0 to 65,544. Adult worms of C. sinensis were collected from 17 egg-positive cases, and the number of worms ranged from 1 to 114 in each individual. A positive correlation between EPGs and numbers of worms was demonstrated (r = 0.681, P<0.001). Worm recovery rates were 9.7% in cases of EPGs 1–1,000 and 73.7% in those of EPGs over 1,000. No worms were detected from egg-negative subjects. Maximum egg count per worm per day was roughly estimated 3,770 in a subject with EPGs 2,664 and 106 collected worms.

Conclusions

The numbers of the worms are significantly correlated with the egg counts in human clonorchiasis. It is estimated that at least 110 worms are infected in a human body with EPGs around 3,000, and egg productivity of a worm per day is around 4,000.     

Risk Factors of Clonorchis sinensis Human Infections in Endemic Areas,Haman-Gun,Republic of Korea: A Case-Control Study

Clonorchis sinensis is the most common fish-borne intestinal parasite in Korea. The aim of the present investigation was to survey the status of C. sinensis infection and analyze associated risk factors in residents of Haman-gun, Gyeongsangnam-do. A total of 5,114 residents from 10 administrative towns/villages voluntarily agreed to participate in the study, which comprised fecal examination, a questionnaire survey for risk factors, ultrasonography, and enzyme-linked immunosorbent assay for cancer biomarker detection in the blood. We detected C. sinensis eggs in 5.3% of the subjects. By region, Gunbuk-myeon had the highest number of residents with C. sinensis eggs. The infection rate and intensity were higher in male than in female residents. Based on the risk factor questionnaire, infection was highly associated with drinking, a history of C. sinensis infection, and the practice of eating of raw freshwater fish. Extension of the bile duct, infection intensity, and cancer biomarker detection significantly correlated with the presence of eggs in the study population. In conclusion, the development of feasible, long-term control policies and strategies for the elimination of C. sinensis in Korea is still required.     

Helminth Eggs Detected in Soil Samples of a Possible Toilet Structure Found at the Capital Area of Ancient Baekje Kingdom of Korea

Although research conducted in East Asia has uncovered parasite eggs from ancient toilets or cesspits, data accumulated to date needs to be supplemented by more archaeoparasitological studies. We examined a total of 21 soil samples from a toilet-like structure at the Hwajisan site, a Baekje-period royal villa, in present-day Korea. At least 4 species of helminth eggs, i.e., Trichuris trichiura, Ascaris lumbricoides, Clonorchis sinensis, and Trichuris sp. (or Trichuris vulpis) were detected in 3 sediment samples of the structure that was likely a toilet used by Baekje nobles. The eggs of T. trichiura were found in all 3 samples (no. 1, 4, and 5); and A. lumbricoides eggs were detected in 2 samples (no. 4 and 5). C. sinensis and T. vulpis-like eggs were found in no. 5 sample. From the findings of this study, we can suppose that the soil-transmitted helminths were prevalent in ancient Korean people, including the nobles of Baekje Kingdom during the 5th to 7th century.     

Two Helminthic Cases of Human Mummy Remains from Joseon-Period Graves in Korea

Our previous research on coprolite specimens from the mummies of Joseon Dynasty (1392–1910 CE) has revealed various species of parasite eggs. Herein, we added 2 new helminthic cases of human remains from Joseon-period graves in the Republic of Korea (Korea). The organic materials precipitated on the hip bones of 2 half-mummied cases (Goryeong and Gwangmyeong cases) were collected, rehydrated, and examined by a microscope. In the sample from Goryeong-gun (gun=County), ova of Trichuris trichiura, Clonorchis sinensis, and Metagonimus spp. were detected, and eggs of T. trichiura and A. lumbricoides were found from the sample of Gwangmyeong-si (si=City). By adding this outcome to the existing data pool, we confirm our previous estimates of Joseon-period parasite infection rates. The overall rates of A. lumbricoides, T. trichiura, and C. sinensis decreased dramatically from Joseon to the modern period. In Goryeong mummy specimen, we also found Metagonimus spp. eggs that has rarely been detected in archaeological samples so far.     

Discovery of Parvatrema duboisi and Parvatrema homoeotecnum (Digenea: Gymnophallidae) from Migratory Birds in Korea

Adult worms of Parvatrema spp. (Digenea: Gymnophallidae) were found in the intestines of 2 species of migratory birds, i.e., a great knot, Calidris tenuirostris, and 2 Mongolian plovers, Charadrius mongolus, in the coastal area of Gunsan-si, Jeollabuk-do in October 2009. The recovered Parvatrema worms were 79 in total number and composed of 2 species. The worms from a great knot were 289 µm in length with the oral and ventral sucker ratio of 2 : 1. They had a single vitellarium, and their intrauterine eggs were 25.0 × 17.5 µm in size. These findings were compatible with P. duboisi (Dollfus, 1923) Bartoli, 1974 (syn. P. timondavidi Bartoli, 1963). The worms recovered from the Mongolian plovers were smaller in length than P. duboisi and had 2 vitellaria. The oral and ventral sucker ratio was 2.5 : 1, and the eggs were 17.5 × 8.8 µm in size. These worms were assigned to be P. homoeotecnum James, 1964. This is the first report on the natural final hosts of Parvatrema spp. in Korea.     

Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends Lifespan in Caenorhabditis elegans

The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants—including slow development, low brood size, and slow defecation—this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.