Screening the Pathogen Box for Identification of New Chemical Agents with Anti-Fasciola hepatica Activity.

Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry. For decades, triclabendazole has been the unique anti-Fasciola drug that can effectively treat this disease. However, triclabendazole resistance in fascioliasis has more recently been reported around the world, and thus, the discovery of novel drugs is an urgent need. The aim of this study was to investigate the fasciocidal properties of 400 compounds contained in the Pathogen Box. The first stage of the screening was carried out by measuring the fasciocidal activity on metacercariae at a concentration of 33 µM each compound (the standard dose). Subsequently, the activities of the most active compounds (n = 33) at their 50% inhibitory concentration (IC50) values against metacercariae were assayed, and the results showed that 13 compounds had IC50s of ≤10 µM. The second stage queried the activities of these compounds at 33 µM against adult flukes, with seven of the compounds producing high mortality rates of >50%. Four hit compounds were selected on the basis of their predicted nontoxic properties, and the IC50 values obtained for adult worms were <10 µM; thus, these compounds represented the best fasciocidal compounds tested here. A cytotoxicity assay on four types of cell lines demonstrated that three compounds were nontoxic at their most active concentration. In conclusion, three hit compounds identified in this proof-of-concept study are potential candidates in the discovery of new fasciocidal drugs. Further studies are warranted.

Effects of miconazole/clotrimazole and praziquantel combinations against the liver fluke Opisthorchis felineus in vivo and in vitro.

The liver fluke Opisthorchis felineus (Rivolta, 1884) is the causative agent of opisthorchiasis felinea in Eurasia. Opisthorchiasis is a serious human and fish-eating animal's disease affecting bile ducts and the gall bladder. Currently, the main drug for specific therapy of opisthorchiasis is praziquantel. We have previously shown that azole inhibitors of O. felineus cytochrome P450 significantly reduced survival of the worms in vitro. Here, we studied in vitro anthelmintic effects of drug combinations involving azole substances approved by the US Food and Drug Administration together with praziquantel against adult or juvenile O. felineus liver flukes. A synergistic interaction was shown for praziquantel-clotrimazole (CI = 0.68) combination and for praziquantel-miconazole (CI = 0.68) combination against adult helminths in vitro. Praziquantel-miconazole (CI = 0.30) had a strongly synergistic effect against newly excysted metacercariae. We also tested anthelmintic effects of azole substances and their combinations with praziquantel in vivo in an animal model of chemotherapy. The treatment of juvenile worms (1 day postinfection) with 100 mg/kg miconazole resulted in a worm burden reduction (WBR) of 37.5% (P = 0.049), with 100 mg/kg clotrimazole causing a WBR of 31.25% (P = 0.025). The treatment of adult worms (5-6 weeks postinfection) with 100 mg/kg or 200 mg/kg miconazole yielded a WBR of 23.8% (P = 0.01) and 21.4% (P = 0.006), respectively. When praziquantel was administered together with clotrimazole or with miconazole, a WBR slightly greater than the effect of ED50 praziquantel was observed (WBR of 59.5 and 54.7%, respectively).In conclusion, the synergistic effect of the praziquantel-clotrimazole and praziquantel-miconazole combinations observed in vitro was not confirmed in vivo. Thus, this combination chemotherapy revealed no benefits over praziquantel monotherapy in the treatment of opisthorchiasis felinea.

Efficacy of injectable praziquantel for elimination of trematode metacercariae in bluegills (Lepomis macrochirus) and quantification of parasite death by propidium iodide staining.

Digenean trematodes have complex life cycles and control of these flatworms can be accomplished by eliminating immature parasite stages from intermediate hosts. In aquaculture systems, presence of trematode metacercariae can negatively impact fish health and lead to economic losses. Posthodiplostomum minimum is a parasite of birds that uses bluegill sunfish (Lepomis macrochirus) as the intermediate host and is commonly found in fish used to stock waterways for recreational purposes. In this study, we evaluated killing of P. minimum metacercariae by injectable praziquantel in naturally infected bluegills. Using propidium iodide staining and motility assessment, we found that 5 mg/kg administered intramuscularly was effective for parasite killing. However, metacercarial death was not apparent until day 7 post-treatment. Our results demonstrated that propidium iodide staining is an effective method for detecting death in metacercariae recovered from treated fish. This method was at least as sensitive as objective motility scoring and provided quantitative assessment of parasite death. Future studies involving treatment of metacercariae in fish with praziquantel may need to be carried out over a period of weeks in order to accurately assess parasite killing and would benefit from using the propidium iodide method.

First report of neurotoxic effect of the cyanobacterium Cylindrospermopsis raciborskii on the motility of trematode metacercariae.

Cylindrospermopsis raciborskii (Woloszynska) is a photosynthetic cyanobacterium that can produce cytotoxic (cylindrospermopsin) and neurotoxic cyanotoxins (saxitoxins). In Brazil the strains of C. raciborskii are reported to produce only saxitoxins (STX) and their effect on fish parasites has not been tested to date. The fish Poecilia vivipara Bloch and Schneider is a common host for the trematode Pygidiopsis macrostomum Travassos off the coast of Rio de Janeiro, and this fish-parasite interaction is a model for behavioural and ecotoxicological studies. The aim of this work was to evaluate the motility of metacercariae of P. macrostomum from P. vivipara exposed to 40 mg l-1 and 400 mg l-1 of crude lyophilized extract of the cyanobacterium C. raciborskii (CYRF-01) for 48 h. The fish were separated into groups of ten individuals and, after exposure, five fish from each group were dissected for counting and checking the motility of metacercariae. The other five fish were dissected after 48 h in clean water. The detection and quantification of STX in the solutions of cyanobacteria, and the gills and guts of fish, were performed by an enzyme-linked immunosorbent assay. The crude extract of C. raciborskii caused temporary paralysis in metacercariae of P. macrostomum after exposure of fish to both concentrations, and the motility recovered after the fish were kept for 48 h in clean water. STX was detected in the guts and gills of all fish analysed, suggesting that this toxin is involved in the paralysis of metacercariae. This is the first report on the action of neurotoxins in metacercariae of fish.

Assessment of in vitro killing assays for detecting praziquantel-induced death in Posthodiplostomum minimum metacercariae.

Control of parasitic infections may be achieved by eliminating developmental stages present within intermediate hosts, thereby disrupting the parasite life cycle. For several trematodes relevant to human and veterinary medicine, this involves targeting the metacercarial stage found in fish intermediate hosts. Treatment of fish with praziquantel is one potential approach for targeting the metacercaria stage. To date, studies investigating praziquantel-induced metacercarial death in fish rely on counting parasites and visually assessing morphology or movement. In this study, we investigate quantitative methods for detecting praziquantel-induced death using a Posthodiplostomum minimum model. Our results revealed that propidium iodide staining accurately identified praziquantel-induced death and the level of staining was proportional to the concentration of praziquantel. In contrast, detection of ATP, resazurin metabolism, and trypan blue staining were poor indicators of metacercarial death. The propidium iodide method offers an advantage over simple visualization of parasite movement and could be used to determine EC50 values relevant for comparison of praziquantel sensitivity or resistance.

Effect of Praziquantel on the Tegument and Digestive Epithelium Ultrastructure of Brachylaima sp. Metacercariae Parasitizing the Edible Land Snail Cornu aspersum.

The edible land snail Cornu aspersum (Pulmonata: Stylommatophora) acts as second intermediate host in the cycle of Brachylaima sp. trematode, harboring free metacercariae in its kidney. The ingestion of undercooked infected snails by humans allows metacercariae to develop to adult stage in the intestine, causing brachylaimiasis. Praziquantel (PZQ) is the drug of choice to treat trematodiasis and it is effective against Brachylaima sp. metacercariae. The objective of this work was to assess, by transmission electron microscopy (TEM), the ultrastructural changes produced on the tegument and gastrodermis of the Brachylaima metacercariae recovered from C. aspersum treated with PZQ in comparison with untreated ones. Snails naturally infected by Brachylaima sp. metacercariae were treated by PZQ both individually and in groups. Metacercariae recovered from treated and control snails were processed for TEM. The tegument of untreated metacercariae was covered by a regular and thick glycocalyx. The syncytial epithelium contained abundant T2 secretory bodies appearing as membrane-bound biconcave disk-vesicles with high electron-dense and uniform content. The T2 secretory bodies located along the external area of the syncytium were mainly arranged at right angles to the apical plasma membrane. In treated metacercariae, the content of the T2 secretory bodies appeared altered, degenerating from high to low electron density, losing its uniform appearance and forming high electron-dense accumulations scattered around the periphery of the vesicle and separated by low electron-dense spaces. The presence of clusters was detectable in the central area. The characteristic arrangement of the T2 secretory bodies observed in untreated metacercariae was lost in treated ones. Vesicles near the apical area of the tegument no longer maintained their arrangement perpendicular to the apical plasma membrane. The characteristic arrangement of T2 secretory bodies and mitochondria was lost. The T2 secretory bodies were also found altered in the tegumental cell bodies, suggesting that the alterations started at the production stage. Mitochondria were severely degenerated and located in the apical area of the tegument. The digestive system displayed a strong contraction, which included the disappearance of the intracecal lumen.

RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis.

Clonorchis sinensis (C. sinensis) infection is still a common public health problem in freshwater fish consumption areas in Asian countries. More molecular evidence are required to speed up the prevention strategies to control this kind of infectious disease. In the present study, to confirm the biological importance of Csenolase followed by our previous observations of the key metabolic enzyme, we explored the RNA silence effect of the Csenolase-derived RNA interference (RNAi) in C. sinensis. The extramembranous region aa105-226 was selected as the target sequence of RNA silence. Csenolase-derived double strand RNA (dsRNA-Csenolase, 366 bp) was synthetized and delivered into C. sinensis by soaking approach. The penetration of dsRNA into adult worms and metacercariae was tracked using fluorescently labeled RNA. Western blotting and qRT-PCR experiments were performed to determine dsRNA-Csenolase-silencing effect. Our results showed that, after incubating for 120 h, dsRNA-Csenolase could effectively target and downregulate the expression of Csenolase in both adult worms (P < 0.001) and metacercariae (P < 0.01), resulting in a remarkable killing effect on C. sinensis adult worms (P < 0.01). Fluorescent Cy3-labeled dsRNA was mostly deposited in the uterus and vitellarium of adult worm and in the cyst wall of metacercaria. The present study is the first report of RNAi trials in C. sinensis, allowing further applications in identifying functional genes in C. sinensis.

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy, morphometry and confocal laser scanning microscopy.

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.

Alcohol and alkalosis enhance excystation of Opisthorchis viverrini metacercariae.

The northeastern region of Thailand has long been known as an endemic area of the human liver fluke infection which is caused by Opisthorchis viverrini. Humans are infected by ingestion of uncooked cyprinoid fish in traditional dishes such as "koi-pla," "pla-som," "pla-jom," and "pla-ra." To date, the prevalence of this parasite infection remains high because of cultural behavior and local beliefs. The popular misunderstanding among people in this area is that alcohol, lemon juice, and fish sauce can kill the parasites. Thus, they believe that they can eat raw fish without the risk of infection. This study attempts to clarify the effects of ethyl alcohol and acidosis-alkalosis on O. viverrini metacercariae excystation. Metacercariae of O. viverrini were obtained from infected cyprinoid fish in a natural reservoir. Most metacercariae were obtained from small cyprinoid fish. Metacercariae were divided into three experimental groups and were treated with solutions containing four different concentrations of ethyl alcohol, four different concentrations of salt, and a range of acidic/basic pH. Metacercariae excystation was observed at the assigned times, and the data were then analyzed. Salt had no effect on excystation. Interestingly, the optimal conditions for O. viverrini excystation were pH 9 and 25 % ethyl alcohol. The present study suggests that raw fish should not be eaten while drinking alcohol or when consuming other ingredients with pH 9, because both alcohol and pH 9 could induce O. viverrini metacercariae excystation, leading to the early development of parasites in the hepatobiliary system.

Comparative effect of mebendazole, albendazole, tribendimidine, and praziquantel in treatment of rats infected with Clonorchis sinensis.

The aim of the study is to understand the anti-Clonorchis sinensis properties of mebendazole and albendazole, and compare to praziquantel and tribendimidine. Two hundred and thirty rats were divided into five batches for experimental treatment. In four batches, each rat was infected orally with 50 or 100 C. sinensis metacercariae. Twenty-eight to 46 days post-infection, groups of rats were treated orally with single doses of mebendazole, albendazole, praziquantel, tribendimidine, or multiple daily doses of albendazole. While in the remaining batch, mebendazole or praziquantel was administered to groups of rats infected each with 50 metacercariae for 7 or 14 days. In each batch of test, untreated but infected rats served as control. All rats were euthanized 2-4 weeks post-drug administration for assessment of efficacy. In the first batch of test, rats treated with mebendazole or tribendimidine at single doses of 150, 75, and 37.5 mg/kg resulted in worm burden reductions of 99.0%, 94.0%, and 73.1%, or 98.0%, 80.6%, and 60.4%, respectively. When rats were treated with albendazole at the same dose levels, no or poor effect was seen. In the second batch of test, promising effect against adult C. sinensis in rats treated with mebendazole or tribendimidine at single doses of 100 and 50 mg/kg were also observed, but under the single dose of 25 mg/kg, only tribendimidine still remained the effect. In the third batch of test, the aforementioned three single dose levels of mebendazole, albendazole and praziquantel were applied. Again, mebendazole exhibited higher effect and albendazole exhibited no or poor effect. While praziquantel, administered at a higher dose of 300 mg/kg, also showed promising effect. In the fourth batch of test, oral administration of albendazole at a daily dose of 150 or 100 mg/kg for 2 or 3 days resulted in moderate or higher efficacy with worm burden reductions of 79.2% and 91.9%, respectively. In the fifth batch of test, single mebendazole doses of 150 or 75 mg/kg exhibited promising effect against 14-day-old C. sinensis in rats with worm burden reductions of 95.3% and 86.4%, respectively, but mebendazole was short of the effect against 7-day-old worms. Under the same dose level, praziquantel possessed an effect against both 7- and 14-day-old juvenile C. sinensis. The results confirm that in infected rats, mebendazole administered orally at a single dose of 150 mg/kg exhibits potential effect against juvenile (14-day-old) and adult C. sinensis. No or less effect is obtained from albendazole under the same dose levels, but extension of treatment course may enhance the effect of albendazole against this species of fluke. The single effective dose ranges of mebendazole and tribendimidine against C. sinensis in rats are similar with a broad window, while the window for praziquantel is narrow.

Viability of the encysted metacercariae of Echinostoma caproni judged by light microscopy versus chemical excystation.

The purpose of this study was to determine the viability of encysted metacercariae of Echinostoma caproni stored in Locke's solution 1:1 at 4 C for 1-24 wk. Viability was judged by light microscopy (LM) based on morphological characteristics of the encysted metacercariae versus chemical excystation of the cysts in a trypsin-bile salts excystation medium. The percent viability was very similar under both methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk poststorage, viability was found to be about 2x greater based on excystation than using LM. We concluded that LM alone underestimated the viability of cysts and that determination of cyst viability was more accurate under assessment by chemical excystation.