Ginkgo biloba attenuated detrimental inflammatory and oxidative events due to Trypanosoma brucei rhodesiense in mice treated with melarsoprol.

BACKGROUND: The severe late stage Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei rhodesiense (T.b.r) is characterized by damage to the blood brain barrier, severe brain inflammation, oxidative stress and organ damage. Melarsoprol (MelB) is currently the only treatment available for this disease. MelB use is limited by its lethal neurotoxicity due to post-treatment reactive encephalopathy. This study sought to assess the potential of Ginkgo biloba (GB), a potent anti-inflammatory and antioxidant, to protect the integrity of the blood brain barrier and ameliorate detrimental inflammatory and oxidative events due to T.b.r in mice treated with MelB. METHODOLOGY: Group one constituted the control; group two was infected with T.b.r; group three was infected with T.b.r and treated with 2.2 mg/kg melarsoprol for 10 days; group four was infected with T.b.r and administered with GB 80 mg/kg for 30 days; group five was given GB 80mg/kg for two weeks before infection with T.b.r, and continued thereafter and group six was infected with T.b.r, administered with GB and treated with MelB. RESULTS: Co-administration of MelB and GB improved the survival rate of infected mice. When administered separately, MelB and GB protected the integrity of the blood brain barrier and improved neurological function in infected mice. Furthermore, the administration of MelB and GB prevented T.b.r-induced microcytic hypochromic anaemia and thrombocytopenia, as well as T.b.r-driven downregulation of total WBCs. Glutathione analysis showed that co-administration of MelB and GB prevented T.b.r-induced oxidative stress in the brain, spleen, heart and lungs. Notably, GB averted peroxidation and oxidant damage by ameliorating T.b.r and MelB-driven elevation of malondialdehyde (MDA) in the brain, kidney and liver. In fact, the co-administered group for the liver, registered the lowest MDA levels for infected mice. T.b.r-driven elevation of serum TNF-α, IFN-γ, uric acid and urea was abrogated by MelB and GB. Co-administration of MelB and GB was most effective in stabilizing TNFα levels. GB attenuated T.b.r and MelB-driven up-regulation of nitrite. CONCLUSION: Utilization of GB as an adjuvant therapy may ameliorate detrimental effects caused by T.b.r infection and MelB toxicity during late stage HAT.

Experimental infection with Toxoplasma gondii in broiler chickens (Gallus domesticus): seroconversion, tissue cyst distribution, and prophylaxis.

Toxoplasma gondii is a widespread zoonotic protozoan that infects most species of mammals and birds, including poultry. This study aimed to investigate the course of T. gondii infection and the efficacy of diclazuril and Artemisia annua in preventing infection in experimentally infected chickens. Seventy-five 1-month-old chickens, female and male, were randomly divided into five groups (n = 15 each) as follows: (1) uninfected untreated (negative control, NC); (2) infected with T. gondii genotype II/III isolated from a wild cat (group WC); (3) infected with T. gondii genotype II isolated from a domestic cat (group DC); (4) infected with T. gondii domestic cat strain and treated with the anticoccidial diclazuril (group DC-D); and (5) infected with T. gondii domestic cat strain and treated with the medicinal plant Artemisia annua (group DC-A). Clinical signs, body temperature, mortality rate, weight gain, feed conversion ratio, hematological parameters, and the presence of T. gondii-specific IgY antibodies were recorded in all groups. Five chickens per group were euthanized 28 days post-infection (p.i.) and their brains, hearts, and breast muscle tested for T. gondii by mouse bioassay and polymerase chain reaction (PCR). No clinical signs related to the experimental infection were observed throughout the study period. T. gondii-specific antibodies were detected by day 28 p.i., but not in all infected chickens. Overall, T. gondii DNA was detected (bioassay or tissue digests) in all infected and untreated chickens (10/10), while viable parasite (bioassay) was isolated from 7 out of 10 chickens. The parasite was most frequently identified in the brain (7/10). There were no differences in the T. gondii strains regarding clinical infection and the rate of T. gondii detection in tissues. However, higher antibody titers were obtained in chickens infected with T. gondii WC strain (1:192) comparing with T. gondii DC strain (1:48). A. annua reduced replication of the parasite in 3 out of 5 chickens, while diclazuril did not. In conclusion, broiler chickens were resistant to clinical toxoplasmosis, irrespective of the strain (domestic or wild cat strain). The herb A. annua presented prophylactic efficacy by reduced parasite replication. However, further studies are required aiming at the efficacy of diclazuril and A. annua for the prevention of T. gondii infection in chickens using quantitative analysis methods.

Curcumin@metal organic frameworks nano-composite for treatment of chronic toxoplasmosis.

Toxoplasmosis is a zoonotic protozoal disease caused by Toxoplasma gondii, an intracellular opportunistic protozoan parasite that can infect any warm-blooded vertebrate cell. In this study, zirconium, and iron-based metal-organic framework was prepared according to the solvothermal method. New nanocomposite (Curcumin@MOFs) was prepared by reacting curcumin with amino-functionalized metal-organic frameworks (Fe-MOF and UiO-66-NH2). Besides characterizations of the composite by powder X-ray diffraction and scanning electron microscope, nano-Curcumin@MOFs was used as a new novel structure as atrial for treatment of chronic toxoplasmosis. Results showed a reduced number of brain cysts, high levels of serum Toxo IgG, and normal histo-morphology with preserved parenchymal, and stromal tissues in rats groups treated with curcumin and Curcumin@MOFs nanocomposite.

Prophylactic Activity of Biogenic Selenium Nanoparticles Against Chronic Toxoplasma gondii Infection.

BACKGROUND: Studies showed that biogenic selenium nanoparticles (SeNPs) have a number of pharmacological properties, such as antimicrobial ones. OBJECTIVE: The present investigation assesses the efficacy of biogenic selenium nanoparticles (SeNPs) as a new patent against latent toxoplasmosis in a mice model. METHODS: Male BALB/c mice were orally treated with SeNPs at the doses of 2.5, 5, 10 mg/kg once a day for 14 days. On the 15th day, the mice were infected with the intraperitoneal inoculation of 20-25 tissue cysts from the Tehran strain of Toxoplasma gondii. The mean numbers of brain tissue cysts and the mRNA levels of TNF-α, IL-12, IL-10, IFN-γ, and inducible nitric oxide synthase (iNOS) in mice of each tested group were measured. Moreover, serum clinical chemistry factors in treated mice were examined to determine the safety of SeNPs. RESULTS: The mean number of the brain tissue cysts was significantly (P<0.001) decreased in mice treated with SeNPs at doses 2.5 (n=37), 5 (n=11), and 10 mg/kg (n=3) based on a dose dependent manner compared with the control group (n=587). The mRNA levels of IFN-γ, TNF-α, IL-12, and iNO were significantly increased in mice treated with SeNPs at the doses 10 mg/kg compared with control subgroups (p<0.05). No significant variation (p>0.05) was observed in the clinical chemistry parameters among the mice in the control subgroups compared with groups treated with SeNPs. CONCLUSION: The results of the present study showed a new patent in the treatment of toxoplasmosis; so that taking the biogenic selenium nanoparticles in concentrations of 2.5-10 mg/kg for 2 weeks was able to prevent severe symptoms of the toxoplasmosis in a mice model. This indicated the prophylactic effects of SeNPs with no considerable toxicity against latent toxoplasmosis. However, more studies are required to elucidate the correct anti-Toxoplasma mechanisms of SeNPs.

Biogenic selenium nanoparticles target chronic toxoplasmosis with minimal cytotoxicity in a mouse model.

Introduction. Nanoparticles (NPs) have numerous biological benefits due to their large surface-volume ratio and convenient entry into cells compared to other particles. Previous research has shown the antimicrobial properties of biogenic selenium NPs (SeNps) and their effects on cellular immunomodulatory cytokines that play a key role in controlling infections.Aim. This study aimed to evaluate the therapeutic effects of SeNPs against chronic toxoplasmosis in mice.Methodology. Infected mice with Toxoplasma gondii (Tehran strain) were orally treated with SeNPs at doses of 2.5, 5 and 10 mg kg-1 once a day for 14 days. On the fifthteenth day, the mean number of brain-tissue cysts and the mRNA levels of TNF-α, IL-12, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) in the mice of each group were recorded. Moreover, serum clinical chemistry factors in the treated mice were examined to determine the safety of SeNPs.Results. The mean number of tissue cysts was significantly (P<0.001) decreased in mice treated with SeNPs in a dose-dependent manner compared with the control group. The mRNA levels of inflammatory cytokines were significantly increased in mice treated with SeNPs at a dose of 10 mg kg-1 compared with the control subgroup (P<0.05). No significant variation (P>0.05) observed in clinical chemistry parameters among the mice in the control subgroup compared with those treated with SeNPs.Conclusion. The findings demonstrated the therapeutic effects of SeNPs with no considerable toxicity against latent toxoplasmosis in the mouse model. Nevertheless, further studies are obligatory to reveal the exact anti-Toxoplasma mechanisms of SeNPs.

Adjunctive bradyzoite-directed therapy for reducing complications of congenital toxoplasmosis.

Congenital toxoplasmosis is caused by in utero infection of the fetus with the intracellular parasite Toxoplasma gondii. Upon infection, the parasite forms life-long cysts in fetal brain and eyes which are resistant to the currently accepted therapy of pyrimethamine and sulfadiazine. These cysts commonly reactivate later in life causing chorioretinitis and visual impairment, and rarely cause neurological complications. I hypothesize that adjunctive, bradyzoite-directed therapies have the potential to alleviate a significant burden of disease by reducing cyst burden in neonatal brain and eyes. Atovaquone is perhaps the most promising drug for further evaluation given its low side-effect profile, established safety, and efficacy in animal models reducing cyst burden. Very limited observational data in humans suggests atovaquone may prevent Toxoplasma-associated chorioretinitis recurrence. Clinical trials are needed to evaluate it and other potential drugs as adjunctive treatment in congenital toxoplasmosis.

Functional Assessment of 2,177 U.S. and International Drugs Identifies the Quinoline Nitroxoline as a Potent Amoebicidal Agent against the Pathogen Balamuthia mandrillaris.

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris-a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions-have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.IMPORTANCEBalamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections.

Could Araucaria heterophylla resin extract be used as a new treatment for toxoplasmosis?

Toxoplasmosis is a worldwide parasitic disease responsible for serious health problems to human. The currently available drugs used for toxoplasmosis treatment showed a limited efficacy and cause serious host toxicity. The in vitro screening for toxoplasmicidal activity of Araucaria heterophylla resin (AHR) extract and its major component 13-epi-cupressic acid (CUP) showed that both AHR (EC50 = 3.90) and CUP (EC50 = 3.69) have high toxoplasmicidal activity in comparison with standard cotrimoxazole (EC50 = 4.28). The antiprotozoal effects of AHR and CUP were investigated against acute and chronic toxoplasmosis using mice models. Two groups of Swiss albino mice were infected by RH Toxoplasma strain intraperitoneally and by Me49 strain orally. Both groups were treated with AHR and CUP in different doses. Their effects were evaluated by survival rate, peritoneal, spleen and liver parasite burdens, brain cyst burden, NO serum level and histopathological lesions. The ultrastructural changes of tachyzoites of acutely infected mice were studied using scanning electron microscopy (SEM). There is an evidence of toxoplasmicidal activity of AHR and CUP in acute and chronic experimental toxoplasmosis. In the acute model, mice treated with AHR and CUP showed prolonged survival rates, a significant decrease in the parasite density in peritoneal lavage and pathological insult in both liver and spleen compared with that of untreated ones. SEM results denote evident morphological alterations of treated tachyzoites. In chronic experimental toxoplasmosis, AHR and CUP treated groups could significantly reduce brain cyst burden by 96.05% and 98.02% respectively. This study indicates that AHR and CUP showed potent toxoplasmicidal activities experimentally and could be used as a potential natural nontoxic agent for treatment of toxoplasmosis.

Development of nanoparticle-assisted PCR assay in the rapid detection of brain-eating amoebae.

Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 µg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 µg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 µg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.

In vitro screening of the open source Pathogen Box identifies novel compounds with profound activities against Neospora caninum.

Neospora caninum is a major cause of abortion in cattle and represents an important veterinary health problem of great economic significance. The Medicines for Malaria Venture (MMV) Pathogen Box, an open-source collection of 400 compounds with proven anti-infective properties against a wide range of pathogens, was screened against a N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts. A primary screening carried out at 1µM yielded 40 compounds that were effective against N. caninum tachyzoites. However, 30 of these compounds also affected the viability of the host cells. The 10 remaining compounds exhibited IC50 values between 4 and 43nM. Three compounds with IC50 values below 10nM, namely MMV676602, MMV688762 and MMV671636, were further characterized in vitro in more detail with respect to inhibition of invasion versus intracellular proliferation, and only MMV671636 had an impact on intracellular proliferation of tachyzoites. This was confirmed by transmission electron microscopy, showing that the primary target of MMV671636 was the mitochondrion. MMV671636 treatment of experimentally infected mice significantly reduced the number of animals with lung and brain infection, and these mice also exhibited a significantly reduced titer of antibodies directed against N. caninum antigens. Thus, MMV671636 is a promising starting point for the development of a future neosporosis therapy.

Effects of Thai piperaceae plant extracts on Neospora caninum infection.

Neosporosis has a worldwide distribution and causes economic losses in farming, particularly by increasing the risk of abortion in cattle. This study investigated the effects of Thai piperaceae (Piper betle, P. nigrum, and P. sarmentosum) extracts on Neospora caninum infections in vitro and in vivo. In an in vitro parasite growth assay based on the green fluorescent protein (GFP) signal, P. betle was the most effective extract at inhibiting parasite growth in human foreskin fibroblast cells (IC50 of GFP-expressing N. caninum parasites, 22.1µg/ml). The P. betle extract, at 25µg per ml, inhibited parasite invasion into host cells. Furthermore, in two independent experiments, treating N. caninum-infected mice with the P. betle extract for 7days post-infection increased their survival. In trial one, the anti-N. caninum effects of the P. betle extract reduced the mouse clinical scores for 30days post-infection (dpi). The survival rate of the mice treated with 400mg/kg was 100% compared with 66.6% for those treated with 100mg/kg and the non-treated controls. In trial two, treating the infected mice with the P. betle extract increased their survival at 50dpi. All mice in the non-treatment group died; however, the survival rates of the 400mg/kg-treated and 100mg/kg-treated mice were 83.3% and 33.3%, respectively. Also, a trend towards a reduced parasite burden was noted in the brains of the P. betle extract-treated mice, compared with the control mice. Therefore P. betle extract has potential as a medicinal plant for treating neosporosis.

Histopathological lesions in encephalon and heart of mice infected with Toxoplasma gondii increase after Lycopodium clavatum 200dH treatment.

In many cases, symptoms of toxoplasmosis are mistaken for the ones of other infectious diseases. Clinical signs are rare in immunocompetent people. However, when they arise, in the acute phase of infection, several organs are affected due to the rapid spread of tachyzoites through the bloodstream. In the present study, the reduction of tachyzoites in peripheral blood of mice of G72 (infected 72h after treatment) and G48 (infected 48h after treatment and treated three more times), when compared with IC (infected and non-treated), suggests protective effect exerted by Lycopodium clavatum. If on the one hand L. clavatum brought benefits, reducing parasitemia, on the other hand, the parasitism became exacerbated. Histopathological analysis demonstrated focal, multifocal and diffuse inflammatory infiltrates, ranging from absent, discreet, moderate to intense, in heart and encephalon of mice of NIC (non-infected and non-treated), IC, G48 and G72 groups, respectively. In the perivascular region and meninges, the injuries were enlarged. The presence of tachyzoites was demonstrated through immunohistochemical (IHC) assay in myocardium. Toxoplasma gondii induced increase of collagen fibers in myocardium of mice of G72 and G48 groups, compared with IC (p<0.05) and NIC (p<0.001). The presence of inflammatory infiltrates, as well as the progressive fibrosis, caused myocardial remodeling in animals treated with L. clavatum. Counterstaining with H&E suggests TGF-ß expression by mononuclear cells in the inflammatory infiltrate. Based on our results, we can conclude that the adopted regimen and potency exerted a protective effect, reducing parasitemia. However, it intensified the histopathological lesions in encephalon and heart of mice infected with T. gondii.

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection.

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine, and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL, or sulfadiazine, and the following parameters analyzed: Cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non-treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model.

Effects of Thymus vulgaris ethanolic extract on chronic toxoplasmosis in a mouse model.

The current work was undertaken to investigate the potential effectiveness of Thymus vulgaris ethanolic extract (TVE) against Toxoplasma gondii infection in chronic experimental toxoplasmosis. To evaluate prophylactic effects, mice received 500 mg/kg TVE for 5 days before they were infected by an avirulent Me49 T. gondii strain. To investigate the therapeutic effects of the extract postinfection, daily treatment with TVE was initiated at 6 weeks postinfection and continued for 10 days. The following groups of animals were used as controls: uninfected/non-treated, infected/non-treated, and infected/treated with a combination of pyrimethamine and sulfadiazine. Brain cyst count and histopathological changes using H&E and Feulgen stains were used to evaluate the efficacy of TVE. The mean number of brain cysts was significantly decreased by 24 % in mice treated prophylactically with TVE. TVE also significantly reduced the mean number of brain cysts when administered to animals already chronically infected with T. gondii. The effect of TVE was comparable to that of treatment with a mixture of sulfadiazine and pyrimethamine (46 and 51 % reduction, respectively). Moreover, considerable amelioration of the pathological lesions in the brain and retina was observed. The results demonstrate the potential efficacy of T. vulgaris as a new natural therapeutic and prophylactic agent for use in the treatment of chronic toxoplasmosis.

Simultaneously targeting inflammatory response and parasite sequestration in brain to treat Experimental Cerebral Malaria.

Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15-20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. Migration of T cells and parasite-infected RBCs (pRBCs) into the brain are both necessary to precipitate the disease. We have been able to simultaneously target both these parameters of ECM. Curcumin alone was able to reverse all the parameters investigated in this study that govern inflammatory responses, CD8(+) T cell and pRBC sequestration into the brain and blood brain barrier (BBB) breakdown. But the animals eventually died of anemia due to parasite build-up in blood. However, arteether-curcumin (AC) combination therapy even after the onset of symptoms provided complete cure. AC treatment is a promising therapeutic option for HCM.

In vivo imaging of trypanosome-brain interactions and development of a rapid screening test for drugs against CNS stage trypanosomiasis.

HUMAN AFRICAN TRYPANOSOMIASIS (HAT) MANIFESTS IN TWO STAGES OF DISEASE: firstly, haemolymphatic, and secondly, an encephalitic phase involving the central nervous system (CNS). New drugs to treat the second-stage disease are urgently needed, yet testing of novel drug candidates is a slow process because the established animal model relies on detecting parasitemia in the blood as late as 180 days after treatment. To expedite compound screening, we have modified the GVR35 strain of Trypanosoma brucei brucei to express luciferase, and have monitored parasite distribution in infected mice following treatment with trypanocidal compounds using serial, non-invasive, bioluminescence imaging. Parasites were detected in the brains of infected mice following treatment with diminazene, a drug which cures stage 1 but not stage 2 disease. Intravital multi-photon microscopy revealed that trypanosomes enter the brain meninges as early as day 5 post-infection but can be killed by diminazene, whereas those that cross the blood-brain barrier and enter the parenchyma by day 21 survived treatment and later caused bloodstream recrudescence. In contrast, all bioluminescent parasites were permanently eliminated by treatment with melarsoprol and DB829, compounds known to cure stage 2 disease. We show that this use of imaging reduces by two thirds the time taken to assess drug efficacy and provides a dual-modal imaging platform for monitoring trypanosome infection in different areas of the brain.

Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection.

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.

Curcumin alleviates eosinophilic meningitis through reduction of eosinophil count following albendazole treatment against Angiostrongylus cantonensis in mice.

Angiostrongylus cantonensis (A. cantonensis) is the most common cause of parasitic eosinophilic meningitis worldwide. By using an animal model of BALB/c mice infected with A. cantonensis, previous studies indicated that the anthelmintic drug, albendazole, could kill A. cantonensis larvae and prevent further infection. However, the dead larvae will induce severe immune responses targeting at brain tissues. To alleviate the detrimental effects caused by the dead larvae, we administered curcumin, a traditional anti-inflammatory agent, as a complementary treatment in addition to albendazole therapy, to determine whether curcumin could be beneficial for treatment. The results showed that although curcumin treatment alone did not reduce worm number, combined treatment by albendazole and curcumin helped to reduce eosinophil count in the cerebrospinal fluid, better than using albendazole alone. This alleviating effect did not affect albendazole treatment alone, since histological analysis showed similar worm eradication with or without addition of curcumin. Nevertheless, curcumin treatment alone and combined albendazole-curcumin treatment did not inhibit MMP-9 expression in the brain tissue. In conclusion, curcumin, when used as a complementary treatment to albendazole, could help to alleviate eosinophilic meningitis through suppression of eosinophil count in the cerebrospinal fluid.

1NM-PP1 treatment of mice infected with Toxoplasma gondii.

Bumped kinase inhibitors (BKIs) target analog-sensitive kinases, which the genomes of mammals rarely encode. Previously, we demonstrated that a BKI effectively suppressed the in vitro replication of Toxoplasma gondii, the causative pathogen of toxoplasmosis, by targeting T. gondii calcium-dependent protein kinase 1 (TgCDPK1) (Eukaryotic Cell, 9: 667-670). Here, we examined whether the BKI 1NM-PP1 reduced parasite replication in vivo. A high dose of 1NM-PP1, by intraperitoneal injection, just before the parasite inoculation effectively reduced the parasite load in the brains, livers, and lungs of T. gondii-infected mice, however, a low dose of 1NM-PP1 with oral administration didn't change the survival rates of infected mice.

Encephalitis caused by Angiostrongylus cantonensis after eating raw frogs mixed with wine as a health supplement.

Angiostrongylus cantonensis also known as the rat lungworm, is prevalent in the Pacific Islands and southeast Asia and is the most common cause of eosinophilic meningitis in humans. Although frogs and toads are known as paratenic hosts of A. cantonensis, they are rarely reported as the infectious source of human angiostrongyliasis. We report a case of encephalitis caused by Angiostrongylus cantonensis after eating raw frogs mixed with wine as a health supplement. Prednisolone at a dose of 1 mg/kg/day was prescribed for 14 days successfully. We advise that travelers and residents of endemic areas should avoid eating raw frogs and a public caution on the danger of eating raw wild animal products or the whole animal is recommended to alleviate such accidental infection.