Establishment and application of unbiased in vitro drug screening assays for the identification of compounds against Echinococcus granulosus sensu stricto.

Echinococcus multilocularis and E. granulosus s.l. are the causative agents of alveolar and cystic echinococcosis, respectively. Drug treatment options for these severe and neglected diseases are limited to benzimidazoles, which are not always efficacious, and adverse side effects are reported. Thus, novel and improved treatments are needed. In this study, the previously established platform for E. multilocularis in vitro drug assessment was adapted to E. granulosus s.s. In a first step, in vitro culture protocols for E. granulosus s.s. were established. This resulted in the generation of large amounts of E. granulosus s.s. metacestode vesicles as well as germinal layer (GL) cells. In vitro culture of these cells formed metacestode vesicles displaying structural characteristics of metacestode cysts generated in vivo. Next, drug susceptibilities of E. multilocularis and E. granulosus s.s. protoscoleces, metacestode vesicles and GL cells were comparatively assessed employing established assays including (i) metacestode vesicle damage marker release assay, (ii) metacestode vesicle viability assay, (iii) GL cell viability assay, and (iv) protoscolex motility assay. The standard drugs albendazole, buparvaquone, mefloquine, MMV665807, monepantel, niclosamide and nitazoxanide were included. MMV665807, niclosamide and nitazoxanide were active against the parasite in all four assays against both species. MMV665807 and monepantel were significantly more active against E. multilocularis metacestode vesicles, while albendazole and nitazoxanide were significantly more active against E. multilocularis GL cells. Albendazole displayed activity against E. multilocularis GL cells, but no effects were seen in albendazole-treated E. granulosus s.s. GL cells within five days. Treatment of protoscoleces with albendazole and monepantel had no impact on motility. Similar results were observed for both species with praziquantel and its enantiomers against protoscoleces. In conclusion, in vitro culture techniques and drug screening methods previously established for E. multilocularis were successfully implemented for E. granulosus s.s., allowing comparisons of drug efficacy between the two species. This study provides in vitro culture techniques for the reliable generation of E. granulosus s.s. metacestode vesicles and GL cell cultures and describes the validation of standardized in vitro drug screening methods for E. granulosus s.s.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

Experimental treatment of cystic echinococcosis: Combination therapy with carvacrol and thymol versus albendazole.

Cystic echinococcosis is a worldwide zoonotic disease caused by Echinococcus granulosus sensu lato (s.l.), which produces serious health and economic problems. For human treatment, chemotherapy with albendazole (ABZ), a derivative of benzimidazoles, is widely used. However, due to its low efficacy and the lack of alternatives to ABZ, novel compounds are urgently needed. Aromatic plants exhibit powerful pharmacological activities, are accessible, have a relatively low cost, and have generally mild toxicities, making them an effective choice to traditional therapies. In particular, the pharmaceutical properties of aromatic plants are partially attributed to essential oils (EOs). The aim of the present study was to assess the in vitro and in vivo effects of the combined carvacrol and thymol against E. granulosus sensu stricto (s.s.). The greatest protoscolicidal effect was observed with the 9:1 and 5:5 (carvacrol:thymol) combinations which caused a marked decrease in viability after 6 days post-incubation, agreeing with the ultrastructural changes obtained. Permeation of the cysts and loss of turgidity was observed with the incubation with the different combinations of carvacrol:thymol. In the clinical efficacy study, the combination of thymol (40 mg/kg) and carvacrol (40 mg/kg) caused a tendency to diminish the weight of the cysts in comparison with the control group. On the other hand, the treatment of infected mice with ABZ, thymol or carvacrol, caused a significant decrease in the weight of the cysts. In conclusion, we here demonstrated the efficacy of different concentrations of combined carvacrol and thymol against E. granulosus s.s. protoscoleces and murine cysts, where short periods of treatment were sufficient to achieve a pharmacological effect. Moreover, we observed a reduction in the weight of the cysts in experimentally infected mice after treatment with carvacrol and thymol. The strategy used has an advantage over synthetic drugs because natural compounds are generally safe and non-toxic. Moreover, the combination of two drugs with different modes of action would cause a reduction in the doses and treatment times. Based on the promising results obtained in vitro, in the future, different doses of the combined drugs will be assayed in vivo to determine the potential of these compounds for the treatment of cystic echinococcosis.

Anti-echinococcal effects of sumac, Rhus coriaria, in a murine model of cystic echinococcosis: Parasitological and molecular evaluation.

Sumac has been traditionally used by people as a medicinal plant for the treatment of different disorders. Cystic echinococcosis (CE) is one of the major zoonotic diseases of human with a worldwide distribution. Long term albendazole therapy is usually associated with side effects including impaired liver function and leukopenia. The present study investigated the efficacy of the methanolic extract of Sumac, Rhus coriaria, on the secondary hydatid cyst development in mice and evaluated sumac effects on the expression of a profile of genes with a potential role in parasite development. Thirty-six mice were intraperitoneally inoculated with of with 3000 protoscoleces and six months after induction of infection were divided into three groups that received either oral sumac extract, albendazole or distilled water. The mice were necropsied 45 days later and the volume and weight of cysts were measured. The expression level of five target genes were analyzed using RT-qPCR. The volume and weight of the cysts were significantly lower in the sumac group compared to the controls. Decreased expressions were found in four out of the five genes following sumac administration. While significantly lower expressions in the sumac group were found for the cdk6, b-raf, fgfr and ras genes, no significant difference was found in cdk2 expression as compared with the control groups. Findings of the present study indicate high efficacy of sumac on the size and volume of secondary hydatid cysts in a murine CE model. Further studies are required to explore the most active and effective ingredients of this natural product.

Efficacy of triple dose albendazole treatment for soil-transmitted helminth infections.

In Malaysia, soil-transmitted helminth (STH) infections still persist among indigenous communities. In the past, local studies have focused mostly on epidemiologic aspects of STH infections with a scarcity of information on the efficacy of deworming treatment. The present study consisted of 2 phases: a cross-sectional phase on current epidemiological status and risk factors of STH infections and a longitudinal study over 6 weeks on triple dose albendazole efficacy against STH infections. A total of 253 participants were recruited at baseline and a pre-tested questionnaire was administered to obtain information on socio-demographics, environmental and behavioural risk factors. Stool samples were evaluated using a modified Kato-Katz technique. Cure rate (CR) and egg reduction rate (ERR) were assessed at 3 weeks following a 3-day course of 400mg albendazole treatment and infection status were observed again at 6 weeks. Baseline positivity of trichuriasis, ascariasis and hookworm infections were 56.1%, 11.9% and 20.2%, respectively. Multivariate analysis showed age below 18 years old (P = 0.004), without latrine in house (P = 0.042) and indiscriminate defecation (P = 0.032) were associated with STH infections. In the longitudinal study (N = 89), CR for trichuriasis was 64.6%, while CR of 100% was observed for both ascariasis and hookworm. ERR was above 90% for all three STH species. A rapid increased of Trichuris trichiura egg output was observed at 6 weeks. In conclusion, STH infections are highly prevalent among indigenous communities. Children and teenagers, poor sanitation and hygiene behaviour were determinants for STH infections. Triple dose albendazole is found to be efficacious against Ascaris lumbricoides and hookworm infections but has moderate curative effect with high ERR against T. trichiura. Although triple dose albendazole regimen has logistic challenges and may not be a routine option, consideration of this treatment regime may still be necessary in selective communities to reduce high intensity of T. trichiura infection.

Ultrastructural tegumental changes of Trichinella spiralis adult and larval stages after in vitro exposure to Allium sativum.

Albendazole (ALB) is the most used therapeutic drug for trichinellosis treatment, but it has many drawbacks. Hence, the search for alternative natural compounds is a goal for researchers. The present work aimed to test the in vitro anthelmintic effect of Tomex (Allium sativum) against adult and muscular larva of Trichinella spiralis (T. spiralis). We incubated adult forms and muscular larvae of T. spiralis with Tomex at concentrations 10, 50, and 100 µg/mL to evaluate the changes that happened on the cuticle using a scanning electron microscopy (SEM). Although ALB was very effective, Tomex also affected motility and the tegumental structure of T. spiralis, which was in proportion to its concentration and incubation time, as Tomex started the mortality of muscular larvae and adult stages after 4 h at 50 µg/mL, and after 6 h at10 µg/mL. A 50% mortality rate of muscular larvae was recorded after 6 h at 100 µg/mL. However, a 50% mortality rate was recorded after 12 h at 10 µg/mL for the adult stage. Adult worms and muscular larvae of T. spiralis incubated with 100 µg/mL of Tomex displayed loss of normal annulations and creases of the cuticle, multiple vesicles, widening of longitudinal furrow space, and multiple minor loss blebs. Our results suggested that Tomex can be a therapeutic agent against adults and larvae stages of T. spiralis.

Effects of Ferula asafetida, closantel, albendazole, oxfendazole, and ivermectin against Haemonchus contortus in goats and sheep.

BACKGROUND: Haemonchosis is a fatal disease of small ruminants caused by the parasite Haemonchus contortus (H. contortus). The most common drugs used in the treatment of H. contortus include albendazole, oxfendazole, and ivermectin. However, as previously reported in the treatment of haemonchosis, these medicines have acquired drug resistance problems over time. Interestingly, natural plant compounds have demonstrated promising effects in the treatment of H. contortus. Therefore, the current study evaluated the effects of plant extract, Ferula asafetida, against common drugs such as albendazole, oxfendazole, ivermectin, and closantel for the treatment of haemonchosis in small ruminants. METHODOLOGY: The current study was conducted on different small ruminant farms in Kasur District, Punjab, Pakistan. The positive animals (n = 720) after coprological examination were selected in this study and divided into two major groups (n = 360 goats and n = 360 sheep). Further, animals were divided into five treatment groups (A-E) and one control group with no treatment (F). Albendazole, oxfendazole, ivermectin, closantel, and Ferula asafetida were administered orally to groups A-E, respectively. The eggs per gram feces (EPG) were determined through the McMaster technique on days 0, 7th, and 14th of treatment. RESULTS: The results showed a significantly higher efficacy of closantel and Ferula asafetida against H. contortus in both goats (100% and 70%; p < 0.05) and sheep (99% and 87%; p < 0.05), respectively. No correlation was observed between EPG reduction with age and gender in both goats and sheep. CONCLUSION: Allopathic drug closantel and herbal drug, Ferula asafetida, have been proved an effective dewormer against H. contortus in small ruminants.

Elimination of Giardia duodenalis BIV in vivo using natural extracts in microbiome and dietary supplements.

In this study, a combination therapy of several natural products was evaluated in vivo in the Giardia duodenalis infection model. G. duodenalis infected mice were treated as follows: distilled water (infected control C+), BIOintestil® (BIO; natural products of Cymbopogon martinii and Zingiber officinale), MicrobiomeX® (MBX; extract of Citrus sinensis and Citrus paradisi), MBX + BIO, Camellia sinensis tea (CPR; black tea). These natural compounds were administered in a dose of 100 mg/day and were compared to G. duodenalis-infected mice treated with albendazole (ALB; 50 mg/Kg/day) and metronidazole (MET; 500 mg/Kg/day), the conventional therapies used to this day. One group remained un-infected and untreated as our control group (C-). Treatment started 8 days after infection, and after 5 days of treatment (7 days for MET), all animals were followed for 15 days. We continuously checked for the presence of G. duodenalis by Faust method, in association with detection of the parasite by PCR from feces, as well for the presence of trophozoites in the intestinal mucosa after sacrifice. Animals treated with MBX, BIO and MBX + BIO presented an undetectable parasitic load until the 15th day of monitoring, while animals treated with CPR, MET and ALB continued to release cysts. Animals in the MBX, MBX + BIO, ALB groups consumed lower feed, MBX, CPR, MET had greater weight and MBX, MBX + BIO, BIO, CPR, C- consumed more water when compared to infected-group control. MBX and BIO alone or associated eliminated G. duodenalis without apparent adverse effects and animals of these groups showed better clinical performance in relation to those with high parasitic load. MET, ALB and CPR only decreased the number of cysts, indicating limitations and therapeutic failure.

Assessment of pharmacological activities of Lygodium microphyllum Cav. leaves in the management of pain, inflammation, pyrexia, diarrhea, and helminths: In vivo, in vitro and in silico approaches.

Lygodium microphyllum Cav. (Family: Lygodiaceae) is a perennial, snake fern and an invasive weed in Florida and also known as old world climbing fern. This study is intended to evaluate the antipyretic, analgesic, anti-inflammatory, antidiarrheal and anthelmintic activity of methanol extract of Lygodium microphyllum Cav. leaves (MELM) by in vivo, in vitro and in silico approaches. In addition, Biovia, PyRx autoDock Vina, UCSF Chimera have been applied to investigate the docking study in order to evaluate the binding interaction and an online tool was used to explore the ADME/T properties of selected bioactive compounds. In acetic acid induced writhing study, MELM inhibited 44.28% and 56.61% of writhes at 200 and 400 (mg/kg) respectively compared to standard drug Diclofenac-Na (10 mg/kg) (74.42% inhibition). In anti-inflammatory experiment by formalin triggered licking method, MELM caused significant (p < 0.05) inhibition of licking in both early phase (42.97%, 63.30%) and late phase (43.35%, 63.03%) at the doses of 200 and 400 mg/kg respectively, whereas reference drug Ibuprofen inhibited paw licking 77.18% in early phase and 76.86% in late phase. MELM also showed promising antipyretic potential where the maximum reduction of fever was produced by MELM 400 mg/kg whose fever lowering capacity is close to the prescribe drug Indomethacin 4 mg/kg, i.p. In Castor oil triggered diarrhea method, MELM delayed the onset time of diarrhea, continuous persistence of wet feces, and decreased the weight of wet feces remarkably. Defection inhibition was achieved 27.56% and 51.72%, for MELM 200 and at 400 (mg/kg) respectively while loperamide 2 (mg/kg) yields 55.17% inhibition of the diarrheal defecation. In anthelmintic bioassay, MELM took 5.83 ± 0.83 and 41.67 ± 1.78 min respectively for paralyzing and death compared to standard drug albendazole; (paralysis time 4.00 ± 0.73 min and death time 31,00 ± 1.71 min). Isoeleutherol, isoquercetin and quercetin were found prominent in molecular docking study and ADME/T analysis verified their drug likeliness. The research validates the moderate analgesic, anti-inflammatory, and remarkable antipyretic, antidiarrheal, anthelmintic activities of the plant extract which can be used an alternative source of novel therapeutics.

Anti-trypanosomal and anthelminthic properties of ethanol and aqueous extracts of Tetrapleura tetraptera Taub.

Trypanosomosis and helminthosis, considered as part of neglected tropical diseases, are parasitic infections of public health importance, especially in Africa. Medicinal plants have been used in most parts of Africa, to treat these parasitic infections. The study aims to determine the anti-trypanosomal and anthelminthic properties of Tetrapleura tetraptera (fruit and stembark). The aqueous extracts of T. tetraptera fruit (TTFaq) and stembark (TTSaq), as well as ethanol extracts of T. tetraptera fruit (TTFe) and stembark (TTSe), were screened for their in vitro anti-trypanosomal and anthelminthic activities against T. b. brucei and Pheretima posthuma worms, respectively. Preliminary phytochemical screening of all extracts and gas chromatography-mass spectrometry (GC-MS) analysis of most active extracts were conducted. TTFaq exhibited anti-trypanosomal activity with IC50 of 18.18 µg/mL. TTSe and TTFe had moderate anti-trypanosomal activity with IC50 of 34.76 and 34.84 µg/mL, respectively. TTSaq had relatively low activity against the parasite with IC50 of 55.03 µg/mL. The SI of T. tetraptera extracts was between the range of 0.14-2.09. TTFaq showed dose-dependent activity causing paralysis and death of the adult worms at all concentrations. At the least concentration of 0.625 mg/mL, TTFaq induced paralysis and death after 101.88 ± 0.8 and 242.64 ± 0.38 min of exposure, respectively compared with the negative control (p < 0.0001). TTFe, TTSe and TTSaq caused paralysis of worms after 318.32 ± 0.74, 422.5 ± 0.72, 422.20 ± 0.55 min of exposure at minimum concentrations of 2.5, 10 and 5 mg/mL, respectively (p < 0.0001). However, no death was observed in worms treated with TTFe, TTSe and TTSaq at all test concentrations. In the presence of sub-minimal inhibitory concentration of the extracts, TTFaq potentiated the anthelminthic activity of albendazole whiles TTFe, TTSaq and TTSe inhibited the activity of albendazole. Phytochemical screening revealed the presence of saponins, triterpenoids, reducing sugars, flavonoids (absent in TTFe), steroids (absent in TTFaq) and tannins (absent in TTSe and TTFe) in the extracts. GC-MS revealed the presence of 9-octadecenamide and betulic acid in TTFaq. Hence, there was evidence provided here that Tetrapleura tetraptera may be effective. This gives credence to their folkloric use. However, further study might be necessary to ascertain safety use in both humans and animals.

The anthelmintic potentials of medicinal plant extracts and an isolated compound (rutin, C27H30O16) from Terminalia catappa L. against Gastrothylax crumenifer.

Paramphistomosis is a pathogenic disease that occurs frequently in tropical and subtropical countries including Thailand. This disease is affected in the parasites causing severe gastrointestinal disorders and death in infected animals. In the present study, we examined the anthelmintic efficacy of albendazole (ABZ) and crude plant extracts from barks of Bombax ceiba L., Diospyros rhodocalyx Kurz. and Vitex glabrata R.Br., and leaves of Terminalia catappa L. and Cassia alata L. against Gastrothylax crumenifer. The hightest anthelmintic activity on the parasites after 24 h incubation was observed in the n-butanol extract of T. catappa leaf. In this study, fractionation bioassay of n-butanol extract of T. catappa leaf was conducted to both separation and discrimination of rutin served as a new efficient compound (LC50 = 28.96; LC90 = 88.75 µg/mL) against G. crumenifer. This compound was confirmed by 1H nuclear magnetic resonance (1H NMR), 13C NMR, infrared (IR) and ultraviolet (UV) spectra as well as mass spectra data. The rutin-treated parasites with all dosages showed swift decrease of the motility and the relative motility (RM) and survival index (SI) were decreased obviously from 3 h until flukes were killed after 12 h of incubation. When observed with light microscopy, the parasites showed the earliest change in a limited region of the tegument. When observed by scanning electron microscopy, the parasites' tegument exhibited similar sequences of surface changes after treatments with rutin and ABZ, but less severity in ABZ treatment. The sequences of changes comprised swelling of folds and ridges, formation of blebbing, rupturing of blebs, erosions, lesions and the tegument demolition. Hence, rutin could be considered as the potential anthelmintic agent for treatment of paramphistomosis.

Copper nanoparticles: Biosynthesis, characterization, and protoscolicidal effects alone and combined with albendazole against hydatid cyst protoscoleces.

BACKGROUND: Surgery remains the preferred treatment option for hydatid cyst (cystic echinococcosis); however, recent studies have demonstrated that the current protoscolicidal agents used during surgery are associated with some adverse side effects such as biliary fibrosis, hepatic necrosis, and cirrhosis. The present study aims to evaluate the in vitro and ex vivo anti-parasitic effects of copper nanoparticles (CuNPs) alone and combined with albendazole on hydatid cyst protoscoleces. METHODS: CuNPs was green synthesized using C. spinosa extract. Various concentrations of CuNPs (250, 500, and 750 mg/mL) alone and combined with albendazole (ALZ, 200 mg/mL) were exposed to protoscoleces collected from the liver fertile hydatid cysts of infected sheep for 5-60 min in vitro and ex vivo. Next, the eosin exclusion test was applied to determine the viability of protoscoleces. Caspase-3 like activity of CuNPs-treated protoscoleces was then evaluated using the colorimetric protease assay Sigma Kit based on the manufacturer's instructions. RESULTS: Scanning electron microscopy (SEM) results showed that the particle size of CuNPs was 17 and 41 nm with the maximum peak at the wavelength of 414 nm. The maximum protoscolicidal activity of CuNPs was observed at the concentration of 750 mg/mL in vitro, so that 73.3 % of protoscoleces were killed after 60 min of exposure. Meanwhile, the mortality of protoscoleces was 100 % after 10 min of exposure to 750 mg/mL of CuNPs along with ALZ (200 mg/mL). Nevertheless, the findings proved that CuNPs even in combination with ALZ required a longer time to kill protoscoleces ex vivo. After 48 h of treating protoscoleces, CuNPs in a dose-dependent manner and at doses of 250, 500, and 750 mg/mL induced the caspase enzyme activation by 20.5 %, 32.3 %, and 36.1 %, respectively. CONCLUSION: The findings of the present investigation showed potent protoscolicidal effects of CuNPs, especially combined with albendazole, which entirely eliminated the parasite after 10-20 min of exposure. The results also showed that although the possible protoscolicidal mechanisms of CuNPs are not clearly understood, the inducing apoptosis through caspases is one of the main protoscolicidal mechanisms of CuNPs. However, supplementary studies, especially in animal models and clinical settings, are needed to approve these results.

Effects of Allium sativum on IFN-γ and IL4 concentrations in mice with cystic echinococcosis.

The aim of the current investigation was to assess the impacts of methanolic extract of Allium sativum (MEAS) on IL-4 (a cytokine derived from Th2 cells) and IFN-É£ (a cytokine derived from Th1 cells) levels in mice infected with Echinococcus granulosus. Sixty healthy BALB/c female mice were used in this study. Each animal was intraperitoneally injected with 1500 protoscoleces. The infected animals were randomly divided into six groups: albendazole (100 mg/kg), MEAS 10 (10 mg/kg), MEAS 20 (20 mg/kg), MEAS 40 (40 mg/kg), MEAS 80 (80 mg/kg) and control group with no treatment. The studied animals received albendazole and/or MEAS through drinking water for 30 days. Serum IFN-γ concentration significantly increased in the MEAS 20 and 80 groups in comparison to the control, albendazole and MEAS 10 groups (P < 0.05). The serum IL-4 level showed no significant difference between the trial groups. The findings of this study showed that MEAS at 20 and 80 mg/kg concentrations enhanced Th1 cell response in mice with cystic echinococcosis.

Drug repurposing applied: Activity of the anti-malarial mefloquine against Echinococcus multilocularis.

The current chemotherapeutical treatment against alveolar echinococcosis relies exclusively on benzimidazoles, which are not parasiticidal and can induce severe toxicity. There are no alternative treatment options. To identify novel drugs with activity against Echinococcus multilocularis metacestodes, researchers have studied potentially interesting drug targets (e.g. the parasite's energy metabolism), and/or adopted drug repurposing approaches by undertaking whole organism screenings. We here focus on drug screening approaches, which utilize an in vitro screening cascade that includes assessment of the drug-induced physical damage of metacestodes, the impact on metacestode viability and the viability of isolated parasite stem cells, structure-activity relationship (SAR) analysis of compound derivatives, and the mode of action. Finally, once in vitro data are indicative for a therapeutic window, the efficacy of selected compounds is assessed in experimentally infected mice. Using this screening cascade, we found that the anti-malarial mefloquine was active against E. multilocularis metacestodes in vitro and in vivo. To shed more light into the mode of action of mefloquine, SAR analysis on mefloquine analogues was performed. E. multilocularis ferritin was identified as a mefloquine-binding protein, but its precise role as a drug target remains to be elucidated. In mice that were infected either intraperitoneally with metacestodes or orally with eggs, oral treatment with mefloquine led to a significant reduction of parasite growth compared to the standard treatment with albendazole. However, mefloquine was not acting parasiticidally. Assessment of mefloquine plasma concentrations in treated mice showed that levels were reached which are close to serum concentrations that are achieved in humans during long-term malaria prophylaxis. Mefloquine might be applied in human AE patients as a salvage treatment. Future studies should focus on other repurposed anti-infective compounds (MMV665807, niclosamide, atovaquone), which showed stronger in vitro activity against E. multilocularis than mefloquine.

Subcutaneous Hydatid Cyst in Laboratory Mice: Is it a Suitable Method for Evaluating Therapeutic Agents against Hydatid Cyst?

Hydatid disease is an economic and public health concern in many countries. Currently, surgery is the main treatment option for hydatid disease. In the surgical treatment of hydatidosis, the use of scolicidal agents is very important due to inactivating live protoscoleces and preventing the recurrence of infection. Therefore, it is necessary to investigate newscolicidal agents and novel medications with higher safety and efficacy. In the previous in vitro studies, the scolicidal effects of the methanolic extracts and aromatic water of Zataria multiflora (Z. multiflora) have been demonstrated. Consequently, in this study, the impact of the nanoemulsion of Z. multiflora essential oil on subcutaneous hydatid cysts was compared with albendazole (ABZ). Fifty laboratory male mice were inoculated with 300 viable protoscoleces subcutaneously on the two sides of the abdomen. Following five months of infection, the remaining infected mice (n=42) were allocated into two treatment and one control (without treatment) groups containing fourteen animals each. Group A received ABZ at the dose of 50 mg/kg for 60 days, group B received the nanoemulsions of Z. multiflora at the dose of 50 mg/kg in drinking water for 60 days, and group C was considered as the control group. All the infected mice were euthanized and necropsied two months post-intervention. Afterwards, the cysts were cautiously collected and their number, size, and weight were compared between the mice of different groups. The mean number of hydatid cysts indicated that the nanoemulsion of Z. multiflora essence had a relative superiority to ABZ. On the other hand, the therapeutic effect of ABZ was higher than the nanoemulsion of Z. multiflora essential oil in terms of the mean weight and mean size of hydatid cysts. However, no significant difference was observed between the groups (P&gt;0.5). Overall, the number, weight, and size of cysts were not significantly different between the groups in this investigation. The lack of satisfactory therapeutic results in this study might be due to the location of hydatid cysts in the subcutaneous space.

Encephalitozoon cuniculi Genotype III Evinces a Resistance to Albendazole Treatment in both Immunodeficient and Immunocompetent Mice.

Of four genotypes of Encephalitozoon cuniculi, E. cuniculi genotype II is considered to represent a parasite that occurs in many host species in a latent asymptomatic form, whereas E. cuniculi genotype III seems to be more aggressive, and infections caused by this strain can lead to the death of even immunocompetent hosts. Although albendazole has been considered suitable for treatment of Encephalitozoon species, its failure in control of E. cuniculi genotype III infection has been reported. This study determined the effect of a 100× recommended daily dose of albendazole on an Encephalitozoon cuniculi genotype III course of infection in immunocompetent and immunodeficient mice and compared the results with those from experiments performed with a lower dose of albendazole and E. cuniculi genotype II. The administration of the regular dose of abendazole during the acute phase of infection reduced the number of affected organs in all strains of mice and absolute counts of spores in screened organs. However, the effect on genotype III was minor. Surprisingly, no substantial effect was recorded after the use of a 100× dose of albendazole, with larger reductions seen only in the number of affected organs and absolute counts of spores in all strains of mice, implying variations in albendazole resistance between these Encephalitozoon cuniculi genotypes. These results imply that differences in the course of infection and the response to treatment depend not only on the immunological status of the host but also on the genotype causing the infection. Understanding how microsporidia survive in hosts despite targeted antimicrosporidial treatment could significantly contribute to research related to human health.

Ultrastructure changes induced by the phloroglucinol derivative agrimol G isolated from Leucosidea sericea in Haemonchus contortus.

Plant extracts used for the treatment of helminth infections in sheep are an alternative to chemical anthelmintic drugs. Previous studies have reported the anthelmintic activity of acetone leaf extracts of Leucosidea sericea. For this study, we evaluate the ultrastructure changes induced by the acetone leaf extract of L. sericea and the component agrimol G (AG) that was isolated for the first time on adult haemonchus parasites. Adult haemonchus parasites harvested from sheep were incubated with the plant extract and AG for 3 h and evaluated by both scanning and transmission electron microscopy in comparison and in combination with albendazole or ivermectin. In all cases the method of evaluation shows ultrastructural changes, with albendazole inducing mitochondrial damage and ivermectin inducing muscle degeneration, both as previously described. Incubation with the plant extract and AG resulted in the formation of numerous non-membrane bound multi-vesicular like bodies and evenly spread disruptions/erosion in the epicuticle. Combining AG with ivermectin or albendazole resulted in an absence of effect of AG. Based on the structural changes induced by AG, together with the absence of an effect in combination with ivermectin and albendazole would suggest a disrupted microtubular network. The latter does however require biochemical confirmation.

Tanshinone IIA attenuates demyelination and promotes remyelination in A. cantonensis-infected BALB/c mice.

BACKGROUND: Angiostrongylus cantonensis infection can cause demyelination in the central nervous system, and there is no effective treatment. METHODS: We used dexamethasone, Tanshinone IIA (TSIIA) and Cryptotanshinone(Two traditional Chinese medicine monomers) in combination with albendazole (AB, a standard anti-helminthic compound) to observe their therapeutic effect on demyelination in A. cantonensis-infected mice. Luxol fast blue staining and electron microscope of myelin sheath, Oligodendrocyte (OL) number and myelin basic protein (MBP) expression in brain was detected in above groups. RESULTS: TSIIA+AB facilitated OL proliferation and significantly increased both myelin sheath thickness and the population of small-diameter axons. In addition, TSIIA treatment inhibited the expression of inflammation-related factors (interleukin [IL]-6, IL-1ß, tumor necrosis factor [TNF]-α, inducible nitric oxide synthase [iNOS]) rather than inhibiting eosinophil infiltration in brain. TSIIA also decreased microglial activation and shifted their phenotype from M1 to M2. CONCLUSIONS: Taken together, these results provide evidence that TSIIA combined with AB may be an effective treatment for demyelination caused by A. cantonensis infection and other demyelinating diseases.

Towards green drugs against cestodes: Effectiveness of Pelargonium roseum and Ferula gummosa essential oils and their main component on Echinococcus granulosus protoscoleces.

Cystic echinococcosis is of great public health importance. Surgery is the efficient treatment for this infection. To minimize the risk of cyst intraoperative leakage, using scolicidals is crucial. To date, any efficacious scolicidal without side effect has not been introduced. Since essential oils of Pelargonium roseum and Ferula gummosa have shown several bioactivities, we evaluated their potential against protoscoleces of E. granulosus using albendazole as positive control. Furthermore, chemical composition of both essential oils was analyzed by gas chromatography-mass spectrometry (GC-MS) analyses, and their main constituents were also evaluated for scolicidal activity. Different concentrations of essential oils and their two main constituents were tested for scolicidal activity. Mortality rate was measured by eosin staining. Results of GC-MS revealed citronellol and ß-pinene as the main constituents of P. roseum and F. gummosa essential oils, respectively. After 60 min of exposure to 50 µg/mL of P. roseum and F. gummosa, mean mortality rate of protoscoleces was 100%. However, ß-pinene and citronellol at the same time point with only 10 µg/mL concentrations resulted in approximately higher than 80% mortality. Essential oils of P. roseum and F. gummosa showed significant toxic effect on E. granulosus with 50% lethal concentration (LC50) values of 8.52 and 17.18 µg/mL, respectively. Based on the LC50 values, ß-pinene (2.20 µg/mL) was the most potent scolicidal agent in the present study. The overall toxicity of ß-pinene and citronellol was significantly higher than the whole essential oils of F. gummosa and P. roseum. Based on these results, ß-pinene and citronellol can be considered as candidate ingredients for the development of green scolicidals.

Allium Sativum Methanolic Extract (garlic) Improves Therapeutic Efficacy of Albendazole Against Hydatid Cyst: In Vivo Study.

After gas chromatography and mass spectrometry of prepared methanolic extract of Allium sativum, 40 laboratory BALB/c mice were infected intraperitoneally by injection of 1,500 viable protoscoleces. Five months after infection, the infected mice were allocated into four treatment groups, including 1- Albendazole (100 mg/kg); 2- Allium sativum methanolic extract (10 mL/L); 3- A. sativum methanolic extract (10 mL/L) + Albendazole (50 mg /kg); and 4- untreated control group. After 30 days of daily treatment, total number and weight of cysts and size of the largest cyst as well as blood serum bilirubin and liver enzymes were compared between the mice of different groups.The total number and weight of cysts and size of the largest cyst were significantly lower in treated groups A. sativum 10 mL/L + Albendazole 50 and Albendazole 100 in comparison to those of the control group (p < 0.05).The activity of alanine aminotransferase (ALT) enzyme and bilirubin concentration were significantly lower in the mice treated with A. sativum 10 mL/L and A. sativum 10 mL/L + Albendazole 50, when compared to the control group. In addition, bilirubin concentration revealed significant decrease in A. sativum 10 mL/L and A. sativum 10 mL/L + Albendazole 50 groups, when compared to the Albendazole group. In conclusion, administration of A. sativum 10 mL/L improved the anti-hydatidosis activity of Albendazole 50 mg /kg, due to parasitological effects similar to Albendazole 100 mg /kg but less hepatotoxic effects.