Parasiticidal Effect Research Articles

Acute lung injury is prevented by monocyte locomotion inhibitory factor in an experimental severe malaria mouse model

Acute lung injury caused by severe malaria (SM) is triggered by a dysregulated immune response towards the infection with Plasmodium parasites. Postmortem analysis of human lungs shows diffuse alveolar damage (DAD), the presence of CD8 lymphocytes, neutrophils, and increased expression of Intercellular Adhesion Molecule 1 (ICAM-1). P. berghei ANKA (PbA) infection in C57BL/6 mice reproduces many SM features, including acute lung injury characterized by DAD, CD8+ T lymphocytes and neutrophils in the lung parenchyma, and tissular expression of proinflammatory cytokines and adhesion molecules, such as IFNγ, TNFα, ICAM, and VCAM. Since this is related to a dysregulated immune response, immunomodulatory agents are proposed to reduce the complications of SM. The monocyte locomotion inhibitory factor (MLIF) is an immunomodulatory pentapeptide isolated from axenic cultures of Entamoeba hystolitica. Thus, we evaluated if the MLIF intraperitoneal (i.p.) treatment prevented SM-induced acute lung injury. The peptide prevented SM without a parasiticidal effect, indicating that its protective effect was related to modifications in the immune response. Furthermore, peripheral CD8+ leukocytes and neutrophil proportions were higher in infected treated mice. However, the treatment prevented DAD, CD8+ cell infiltration into the pulmonary tissue and downregulated IFNγ. Moreover, VCAM-1 expression was abrogated. These results indicate that the MLIF treatment downregulated adhesion molecule expression, impeding cell migration and proinflammatory cytokine tissular production, preventing acute lung injury induced by SM. Our findings represent a potential novel strategy to avoid this complication in various events where a dysregulated immune response triggers lung injury.

Potential i-Nos/Arg-1 Switch with NLRP3 and Parasitic Load Down Regulation in Experimental Schistosoma mansoni Infection via Chloroquine Repurposing.

In previous studies, the inhibitory effect of chloroquine on NLRP3 inflammasome and heme production was documented. This may be employed as a double-bladed sword in schistosomiasis (anti-inflammatory and parasiticidal). In this study, chloroquine's impact on schistosomiasis mansoni was investigated. The parasitic load (worm/egg counts and reproductive capacity index [RCI]), i-Nos/Arg-1 expression, splenomegaly, hepatic insult and NLRP3-immunohistochemical expression were assessed in infected mice after receiving early and late repeated doses of chloroquine alone or dually with praziquantel. By early treatment, the least RCI was reported in dually treated mice (41.48 ± 28.58) with a significant reduction in worm/egg counts (3.50 ± 1.29/2550 ± 479.58), compared with either drug alone. A marked reduction in the splenic index was achieved by prolonged chloroquine administration (alone: 43.15 ± 5.67, dually: 36.03 ± 5.27), with significantly less fibrosis (15 ± 3.37, 14.25 ± 2.22) than after praziquantel alone (20.5 ± 2.65). Regarding inflammation, despite the praziquantel-induced significant decrease in NLRP3 expression, the inhibitory effect was marked after dual and chloroquine administration (liver: 3.13 ± 1.21/3.45 ± 1.23, spleen: 5.7 ± 1.6/4.63 ± 2.41). i-Nos RNA peaked with early/late chloroquine administration (liver: 68.53 ± 1.8/57.78 ± 7.14, spleen: 63.22 ± 2.06/62.5 ± 3.05). High i-Nos echoed with a parasiticidal and hepatoprotective effect and may indicate macrophage-1 polarisation. On the flip side, the chloroquine-induced low Arg-1 seemed to abate immune tolerance and probably macrophage-2 polarisation. Collectively, chloroquine synergised the praziquantel-schistosomicidal effect and minimised tissue inflammation, splenomegaly and hepatic fibrosis.

Use of Medicinal Plants Against Parasitic Infections

Many of medicinal plants are called "ethnobotanicals" used in traditional homemade remedies that have been used for centuries in various regions. Some of the chemicals in plant extracts are well known and characterized (e.g. azadirachtin, citronella, nicotine, etc.) and can be considered as "natural" active ingredients. Some of these active ingredients have genuine insecticidal or anthelmintic properties (i.e. they actually kill some parasites). They are also known as biopesticides. Many of them act as repellent whereas some of them act as insecticide. Herb mixtures (dried, powdered, pelleted, etc.) are commercially available for preparing infusions or for direct feeding to livestock or pets, with various claims against external or internal parasites. Industrially produced plant extracts (e.g. citronella oil and neem oil) are usually well characterized and standardized (Benavides et al., 2001; Ismail et al., 2002 and John et al. 2009). A common feature of most of these natural products is that they are not persistent. They are easily degradable (e.g. by sunlight) and/or rather volatile, or quickly metabolized and excreted, which are rather positive features regarding safety. For parasiticidal effect treatment has to be repeated frequently for constant protection of animals against parasites. Use of these herbal products against parasitic infections becomes mandatory owing to development of resistance against commonly used chemical anti-parasitic compounds. As the anti-parasitic resistance is the genetic ability of parasites to survive treatment with an anti-parasitic drug that was generally effective against those parasites in the past. After treatment with an anti-parasitic drug, the susceptible parasites die and the resistant parasites survive to pass on resistance genes to their offspring.

Stationary metal sheets (copper, zinc or brass) in fish tanks prevent Ichthyophthirius multifiliis Fouquet, 1876 infection of rainbow trout: In vivo and in vitro effects

Infections with the parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 challenge health of freshwater fishes worldwide by eliciting white spot disease (ichthyophthiriosis). A series of chemicals, pharmaceuticals and herbal extracts are currently in use to control the infections by regular or continuous administration to aquaculture systems. Recently application of brass (copper/zinc alloys) metal sheets in marine fish tanks was shown to eliminate tomonts of a related ciliate, Cryptocaryon irritans Brown, 1951 causing cryptocaryonosis (marine white spot disease). The present work documents that brass, and each of its pure constituents (copper and zinc), have in vivo and in vitro antiparasitic effects in freshwater. We used an experimental model with I. multifiliis infected rainbow trout (Oncorhynchus mykiss Walbaum, 1792). In the in vivo study metal sheets of either brass (alloy of copper and zinc), pure copper or pure zinc, were placed in fish tanks with trout experimentally infected with I. multifiliis (duplicated trials). All three metals (pure or combined) effectively blocked the life cycle of I. multifiliis and inhibited reinfection and morbidity. In control tanks with infected trout, but no metal sheets, we observed significantly elevated infection levels and development of disease. In vitro assays, exposing three life cycle stages (tomont, tomocyst and theront) to the same metal sheets in Petri dishes, confirmed the antiparasitic effects. Tomonts were sensitive to pure copper and brass (100% and 66% mortality within 15 min, respectively) and zinc (100% mortality within 30 min). Tomocysts, with enclosed tomites, were less sensitive, as even 12 h exposure to brass, pure copper and zinc merely led to mortality rates of 44%, 50% and 60%, respectively. Theronts were killed within 75 min when exposed to brass and pure copper plates, whereas zinc plate exposure eliminated all theronts within 60 min. Microscopical observations suggested that direct contact between the parasite and the metal sheet increased the parasiticidal effect, although the released metal ions may contribute may play a role. The use of stationary metal sheets in aquaculture settings may be a possible way to control ichthyophthiriosis. However, we measured the concentration of free metal ions in the fish tanks with metal sheets, which suggested release of metal to the water. This calls for additional studies on toxicology and environmental effects of metal sheet use before implementation at fish farms level.

In Vitro versus in Mice: Efficacy and Safety of Decoquinate and Quinoline-O-Carbamate Derivatives against Experimental Infection with Neospora caninum Tachyzoites.

The effects of decoquinate (DCQ) and three O-quinoline-carbamate-derivatives were investigated using human foreskin fibroblasts (HFF) infected with Neospora caninum tachyzoites. These compounds exhibited half-maximal proliferation inhibition (IC50s) from 1.7 (RMB060) to 60 nM (RMB055). Conversely, when applied at 5 (DCQ, RMB054) or 10µM (RMB055, RMB060), HFF viability was not affected. Treatments of infected cell cultures at 0.5µM altered the ultrastructure of the parasite mitochondrion and cytoplasm within 24 h, most pronounced for RMB060, and DCQ, RMB054 and RMB060 did not impair the viability of splenocytes from naïve mice. Long-term treatments of N. caninum-infected HFF monolayers with 0.5µM of each compound showed that only exposure to RMB060 over a period of six consecutive days had a parasiticidal effect, while the other compounds were not able to kill all tachyzoites in vitro. Thus, DCQ and RMB060 were comparatively assessed in the pregnant neosporosis mouse model. The oral application of these compounds suspended in corn oil at 10 mg/kg/day for 5 d resulted in a decreased fertility rate and litter size in the DCQ group, whereas reproductive parameters were not altered by RMB060 treatment. However, both compounds failed to protect mice from cerebral infection and did not prevent vertical transmission/pup mortality. Thus, despite the promising in vitro efficacy and safety characteristics of DCQ and DCQ-derivatives, proof of concept for activity against neosporosis could not be demonstrated in the murine model.

Effects of rusted copper alloy in prevention and control of cryptocaryoniasis in large yellow croaker Larimichthys crocea

Copper alloy can be used to prevent and control cryptocaryoniasis in marine fish. However, it is not known whether the soaking corrosion of seawater can affect the parasiticidal effect of copper alloy. This study compared the killing effects of copper alloy mesh (CAM) and rust copper alloy mesh (RCAM) on the protomonts and tomonts of Cryptocaryon irritans and the control effects of cryptocaryoniasis in Larimichthys crocea. Results showed that with the increase of contact time with CAM and RCAM, the survival rate of the protomonts and tomonts of C. irritans were gradually decreased. The contents of Cu2+ and malondialdehyde (MDA) in the tomonts gradually increased, while the activities of superoxide dismutase (SOD), Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the tomonts increased first and then decreased. Morphological observation showed that sufficient contact with CAM or RCAM damaged the cyst wall of the tomonts, and the cytoplasm have lysed and spilled out of the cell. In the treatment test of diseased fish, at the end of the assay (14 d), the survival rates of L. crocea in the CAM treatment group (group B) and the RCAM treatment group (group C) were 77.77% and 64.44%, respectively, which were significantly higher than those in the untreated group (group A, 0.00%) (P < 0.05). RCAM could still successfully prevent the outbreak of cryptocaryoniasis in a short time. Our study serves as a theoretical reference for the long–term application of metal materials in the prevention and control of cryptocaryoniasis in marine fish.

Isolation and Characterization of NpCI, a New Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita peloronta with Anti-Plasmodium falciparum Activity

Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1·× 10-8 mol/L) and porcine CPB (Ki = 8.15·× 10-8 mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC50 = 5.5 μmol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite's intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite's life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies.

In vitro protoscolicidal effects of lithocholic acid on protoscoleces of Echinococcus granulosus and its mechanism

Surgery has been found to be the best choice of treatment for hydatidosis. However, leakage of cyst contents during surgery is the foremost reason for recurrence of hydatidosis. In this study, we investigated the in vitro efficacy of lithocholic acid (LCA) against Echinococcus granulosus protoscoleces. The protoscoleces were divided into a control group, an albendazole (ABZ) positive control group and LCA intervention groups at concentrations of 0.5, 1, 2, and 3 mmol/L and stained with 0.1% eosin for observation using an inverted microscope; the protoscolecal ultrastructure was examined with SEM and TEM; the activities of ROS, SOD, and caspase-3 were investigated using an ROS kit, SOD kit, and caspase-3 kit, respectively; the contents of HO-1 and NQO-1 were analyzed by enzyme-linked immunosorbent assay; and the expression level of cytochrome c (Ctyc) was analyzed by western blotting. Results: As the concentration of LCA increased, the survival rate of protoscoleces gradually decreased. The microstructure shows that the external shape and internal structure were gradually deformed and collapse. SOD, GSH, HO-1 and NQO-1 decreased more significantly in the 3 mmol/L LCA group. However, ROS levels gradually increased. LCA treatment for 3 days at all concentrations significantly increased caspase-3 activity and expression in a dose-dependent manner. LCA decreased the level of Ctyc protein in vitro. LCA demonstrated a parasiticidal effect on the protoscoleces of Echinococcus granulosus in vitro. LCA may induce apoptosis of E. granulosus protoscoleces by oxidative stress and mitochondrial pathways.

Benznidazole Anti-Inflammatory Effects in Murine Cardiomyocytes and Macrophages Are Mediated by Class I PI3Kδ.

Benznidazole (Bzl), the drug of choice in many countries for the treatment of Chagas disease, leads to parasite clearance in the early stages of infection and contributes to immunomodulation. In addition to its parasiticidal effect, Bzl inhibits the NF-κB pathway. In this regard, we have previously described that this occurs through IL-10/STAT3/SOCS3 pathway. PI3K pathway is involved in the regulation of the immune system by inhibiting NF-κB pathway through STAT3. In this work, the participation of PI3K in the immunomodulatory effects of Bzl in cardiac and immune cells, the main targets of Chagas disease, was further studied. For that, we use a murine primary cardiomyocyte culture and a monocyte/macrophage cell line (RAW 264.7), stimulated with LPS in presence of LY294002, an inhibitor of PI3K. Under these conditions, Bzl could neither increase SOCS3 expression nor inhibit the NOS2 mRNA expression and the release of NOx, both in cardiomyocytes and macrophages. Macrophages are crucial in the development of Chronic Chagas Cardiomyopathy. Thus, to deepen our understanding of how Bzl acts, the expression profile of M1-M2 macrophage markers was evaluated. Bzl inhibited the release of NOx (M1 marker) and increased the expression of Arginase I (M2 marker) and a negative correlation was found between them. Besides, LPS increased the expression of pro-inflammatory cytokines. Bzl treatment not only inhibited this effect but also increased the expression of typical M2-macrophage markers like Mannose Receptor, TGF-β, and VEGF-A. Moreover, Bzl increased the expression of PPAR-γ and PPAR-α, known as key regulators of macrophage polarization. PI3K directly regulates M1-to-M2 macrophage polarization. Since p110δ, catalytic subunit of PI3Kδ, is highly expressed in immune cells, experiments were carried out in presence of CAL-101, a specific inhibitor of this subunit. Under this condition, Bzl could neither increase SOCS3 expression nor inhibit NF-κB pathway. Moreover, Bzl not only failed to inhibit the expression of pro-inflammatory cytokines (M1 markers) but also could not increase M2 markers. Taken together these results demonstrate, for the first time, that the anti-inflammatory effect of Bzl depends on PI3K activity in a cell line of murine macrophages and in primary culture of neonatal cardiomyocytes. Furthermore, Bzl-mediated increase expression of M2-macrophage markers involves the participation of the p110δ catalytic subunit of PI3Kδ.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Trypanocidal therapy among children infected by Trypanosoma cruzi. Serological and electrocardiographic changes over a mean twenty-five-years follow-up period

This study compared the serological and electrocardiographic evolution among patients with chronic T. cruzi infection treated during childhood or left untreated. A retrospective cohort study was conducted during a mean follow-up period of 25 years in 82 patients: half of them underwent treatment (nifurtimox 8, benznidazole 33) before being 15 years old, whereas the other half remained untreated. During the follow-up, negative seroconversion occurred in 92.7% of the treated children, while all the untreated ones remained positive for conventional serology. At baseline, 2 patients from each group had electrocardiographic abnormalities. During the study period, 4/41 (9.75%) and 9/41 (21.95%) of treated and untreated patients displayed an altered electrocardiogram, respectively. In multivariate analyses, the probability of developing electrocardiographic abnormalities was significantly reduced among treated patients (OR = 0.18, 95% CI = 0.04-0.79; p = 0.023). Electrocardiographic abnormalities attributable to Chagas cardiomyopathy were seen in 3 patients from the untreated group (complete right bundle branch block + left anterior fascicular block, frequent ventricular extrasystole, and left anterior fascicular block). The remarkable seronegativization seen in Benznidazole and Nifurtimox recipients underlines the parasiticidal effect of both compounds. Such demonstration along with the fact that CCC-related alterations were only present in the untreated group, reinforces the view of trypanocidal treatment in chronically T. cruzi-infected children as decreasing the risk for cardiomyopathy development.

In vivo and in vitro efficacy of crocin against Echinococcus multilocularis

BackgroundAlveolar echinococcosis (AE) is a fatal zoonosis caused by the larvae of Echinococcus multilocularis. However, current chemotherapy treatment options are based on benzimidazoles [albendazole (ABZ) and mebendazole], which have limited efficacy. Therefore, novel drugs are necessary for the treatment of this disease.MethodsThe anthelmintic effects of crocin were tested on E. multilocularis metacestodes, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFFs) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. The in vivo efficacy of crocin was investigated in mice following secondary infection with E. multilocularis. Furthermore, collagen deposition and degradation in host tissues around the metacestodes were evaluated.ResultsIn vitro, crocin had a median effective concentration of 11.36 μM against cultured E. multilocularis metacestodes, while it reduced germinal cell viability at a median inhibitory concentration of 10.05 μM. Crocin was less toxic to HFFs and RH mammalian cell lines than to metacestodes. Transmission electron microscopy revealed that crocin treatment resulted in structural damage in the germinal layer. In addition, 60.33 ± 3.06% of protoscoleces were killed by treatment with 10 μM crocin for 7 days, indicating that crocin has a parasiticidal effect. In vivo, the metacestode weight was significantly reduced after the administration of crocin at 50 mg/kg and 100 mg/kg (55.1 and 68.1%, respectively). Metacestode pathology showed structural disruption of the germinal and laminated layers after crocin treatment. The crocin- and ABZ-treated groups presented significant increases in the levels of interleukin (IL)-2 and IL-4. Furthermore, crocin inhibited the expression of matrix metalloproteinases (MMPs) (MMP2 and MMP9) and promoted collagen deposition in the metacestode.ConclusionsCrocin was demonstrated to exert parasiticidal activity against E. multilocularis in vitro and in vivo, and can be developed as a novel drug for the treatment of AE.Graphical abstract

Comparative In Vitro and In Vivo Effects of Feed Additives on Rainbow Trout Response toIchthyophthirius multifiliis

AbstractControl of the skin parasitic ciliateIchthyophthirius multifiliisis currently based on laborious chemical and mechanical approaches, thus advocating for alternative control measures. Here, we show that the early development of trophonts (feeding stage residing in the epidermis) in the Rainbow TroutOncorhynchus mykissepidermis was inhibited at 5 d postinfection after the administration of feed additives, including garlicAllium sativum, oreganoOriganum onites, thymeThymus vulgaris, corianderCoriandrum sativum, and astaxanthin (a carotenoid derived from the algaHaematococcus pluvialis). However, no inhibition was observed at 8 d postinfection. We investigated whether the early inhibition was caused by (1) the feed additives’ direct effect on the parasite and/or (2) stimulation of the Rainbow Trout immune responses. Garlic exhibited the highest in vitro killing capacity toward theronts (the infective stage of the parasite), followed by oregano, thyme, and astaxanthin, whereas coriander had no in vitro parasiticidal effect. Immune reactions were measured by plasma lysozyme activity in Rainbow Trout after feeding and by recording immune gene expression in trout leukocytes that were exposed to feed additives. Oregano‐fed fish showed a significantly (P &lt; 0.05) elevated plasma lysozyme activity. Oregano and thyme—and to a lesser extent, garlic, astaxanthin, and coriander—induced a significant regulation of innate and adaptive immune genes in vitro. The results suggest that the investigated additives inhibit early parasite development directly as well as indirectly (i.e., by elevating the host immune response).

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model

BackgroundEchinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice.MethodsThe anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction.ResultsIn vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 μg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm.ConclusionsVepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.Graphical

Eimeria bateri Natural Infection: Oocysts Reductions in Grey Quail (Coturnix coturnix) Treated with Bacillus thuringensis var. israelensis

HIGHLIGHTS Coccidiosis cause important economic losses for poultry industry. Bti produce proteins with high specific parasiticidal effect. Bti reduce the number of Eimeria bateri oocysts in quail.

Enhanced Antimalarial Efficacy Obtained by Targeted Delivery of Artemisinin in Heparin-Coated Magnetic Hollow Mesoporous Nanoparticles.

Malaria is one of the deadliest infectious diseases threatening half of the world population. With the deterioration of the parasiticidal effect of the current antimalarials, novel approaches such as screening of more specific inhibitors and targeted delivery of drugs have been under intensive research. Herein, we prepare hollow mesoporous ferrite nanoparticles (HMFNs) of 200 nm with ferromagnetic properties using a one-pot hydrothermal reaction. A magnetically targeted drug-delivery system coloaded with artemisinin in the inner magnetite shell and heparin on the outer mesoporous shell (HMFN@ART@HEP) is developed. Specific targeting of the magnetic nanoparticles to the parasite-infected erythrocytes is achieved by the attraction between the HMFNs and hemozoin (paramagnetic), a vital metabolite of plasmodium in the erythrocytic stage. With the hemozoin production reaching the maximum during the schizont period of the parasite, HMFN@ART@HEPs are adsorbed to the infected red blood cells (iRBCs), which not only interferes with the release of merozoites but also significantly enhances the inhibitory efficacy due to the increased local concentration of artemisinin. Subsequently, the heparin coated on the surface of the nanoparticles can efficiently interfere with the invasion of freshly released merozoites to new RBCs through the specific interaction between the parasite-derived ligands and heparin, which further increases the inhibitory effect on malaria. As a cluster of heparin, heparin-coated nanoparticles provide stronger blocking capability than free heparin, resulting from multivalent interactions with surface receptors on merozoite. Thus, we have developed a HMFN-based delivery system with considerable antimalarial efficacy, which is a promising platform for treatment against malaria.

Anti-parasitic Activity of Some Medicinal Plants Essential Oils on Giardia lamblia and Entamoeba histolytica, In Vitro

Background and objectives: Giardiasis and amoebiasis are two common diseases in human societies which make increasing problems for the health managing systems. In the present study, the antiparasitic activity of Allium sativum, Artemisia sieberi, Zatraria multiflora, Chenopodium botrys and Eucalyptus globulus essential oils on Giardia lamblia cysts and Entamoeba histolytica trophozoites were investigated in vitro. Methods: Giardia lamblia cysts and E. histolytica trophozoites were isolated from infected sample stools. The parasites were treated by four concentrations (0.2, 0.1, 0.01 and 0.001 µg/mL) of essential oils, separately for 30 min at 37 °C. Results: Theessential oils at the concentration of 0.1 and 0.2 µg/mL indicated appreciate parasiticidal effect (p <0.05). Eucalyptus globulus had maximum efficacy on both G. lamblia and E. histolytica with mortality rates of 79.7% and 87.6% within 30 min. Conclusion: All essential oils showed potent anti-giardiasis and anti-amoebiasis activity. Also, E. globulus with the highest efficiency could be considered as anti-protozoa medication to use an adjunct or primary therapy.

Effect of nutraceuticals on acanthocephalan Neoechinorhynchus buttnerae and its toxicity to the host tambaqui Colossoma macropomum.

The production of tambaqui Colossoma macropomum has been undergoing financial losses due to parasitic infection by the acanthocephalan Neoechinorhynchus buttnerae, raising an alert for aquaculture in South America. The lack of adequate treatment and use of unlicensed chemicals encourages research for alternative solutions with minimal side effects. The objectives of this study were to evaluate the in vitro antiparasitic potential of commercial nutraceutical products (Natumix® and BioFish®) against N. buttnerae and to assess the respective in vivo toxic effects on the host tambaqui. For in vitro assays, parasitized fish were necropsied for acanthocephalans sampling. The parasites were exposed to three concentrations (0.078, 0.313 and 1.25 mg/ml) of each product, as well as controls (one without product and another with a solubilizer). For the in vivo acute toxicity test, juvenile fish (<0.1 g) were exposed to five increasing concentrations of each product. Mortality of tambaqui was recorded at 24, 48, 72 and 96 h. The estimated lethal concentration (LC) for 10, 50, 90 and 99% of fish was determined to classify the toxicity of the products on the target species. After in vitro efficacy tests, the highest concentrations (1.25 mg/ml) caused 100% mortality of the parasites in both products, but only Natumix® caused 100% mortality using the intermediate concentration (0.313 mg/ml) after 24 h. According to the acute toxicity result, the LC50 classified the nutraceutical products as slightly toxic for tambaqui. The tested products had a parasiticidal effect on N. buttnerae, and the toxicity test showed that both products have therapeutic potential when added to the diet.

In vitro efficacies of solubility-improved mebendazole derivatives against Echinococcus multilocularis.

Recently, we introduced an epoxy group to mebendazole by a reaction with epichlorohydrin and obtained two isoforms, mebendazole C1 (M-C1) and mebendazole C2 (M-C2). The in vitro effects of mebendazole derivatives at different concentrations on Echinococcus multilocularis protoscoleces and metacestodes as well as cytotoxicity in rat hepatoma (RH) cells were examined. The results demonstrated that the solubility of the two derivatives was greatly improved compared to mebendazole. The mortality of protoscoleces in vitro reached to 70-80% after 7 days of exposure to mebendazole or M-C2, and M-C2 showed higher parasiticidal effects than mebendazole (P > 0.05). The parasiticidal effect of M-C1 was low, even at a concentration of 30 µm. The percentage of damaged metacestodes that were treated with mebendazole and M-C2 in vitro at different concentrations were similar, and M-C1 exhibited insignificant effects on metacestodes. Significant morphological changes on protoscoleces and metacestodes were observed after treatment with mebendazole and M-C2. In addition, the introduction of an epoxy group to mebendazole also reduced its cytotoxicity in RH cells. Our results demonstrate that the introduction of an epoxy group not only improved the solubility of mebendazole, but also increased its parasiticidal effects on E. multilocularis and reduced its cytotoxicity in RH cells.

In vitro therapeutic effect of Hemiscorpius lepturus venom on tachyzoites of Toxoplasma gondii.

Pyrimethamine which is a main anti-Toxoplasma gondii drug has a serious side and toxic effects on the host. Accordingly, the development of new treatment options for toxoplasmosis with less toxic effects, low teratogenicity and parasiticidal effect against the various stage of T. gondii are dramatically crucial. Currently, natural molecules from scorpion and snake venoms are widely used as an alternative treatment against human disease, these compounds considered to be safe and to have low toxicity in comparison with synthetic drugs. Therefore, the goal of our study was to investigate the anti-Toxoplasma gondii activities of Hemiscorpius lepturus venom. We measured cytotoxicity of H. lepturus whole venom on Vero cells as well as effectiveness of this compound on viability of T. gondii applying colorimetric assay, according to mitochondrial oxidation of the MTT reagent (Methylthiazol tetrazolium 98%). The results of this study indicated that the H. lepturus whole venom has an anti-Toxoplasma effects with less toxic effect on Vero cells. Also, the T. gondii tachyzoites were treated with H. lepturus venom reached better results in comparison with Pyrimethamine-treated group. This research will serve as a base for future studies on toxoplasmosis and suggest a role for scorpion venom in promoting natural drugs.