Heat Shock Protein 90 as a Drug Target against Protozoan Infections Biochemical characterization of HSP90 From plasmodium falciparum and trypanosoma evansi and evaluation of its inhibitor as a candidate drug

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Intestinal protozoan parasites with zoonotic potential in birds

The aim of this study was to evaluate the occurrence of potentially zoonotic intestinal protozoan infections in exotic and wildlife Brazilian birds. Fecal samples from 207 birds of 45 species were examined. Infections by Balantidium sp., Entamoeba sp., and Blastocystis sp. were observed in 17 individuals (8.2%) of Gnorimopsar chopi, Oryzoborus angolensis, Sporophila caerulescens, Ramphastos toco, Aratinga leucophtalmus, and Pavo cristatus.

A coprological survey of parasites of wild mantled howling monkeys, Alouatta palliata palliata.

Fecal samples from 155 mantled howling monkeys (Alouatta palliata palliata) examined at Centro Ecologico La Pacifica, Guanacaste Province, Costa Rica, revealed 75 (48%) had parasitic infections. A sampling of nine howling monkeys from Santa Rosa National Park. Costa Rica indicated only one infected animal (11%). Only three of 19 (16%) spider monkeys (Ateles geoffroyi) also from Santa Rosa were infected. Controrchis biliophilus, Trypanoxyuris minutus, unidentified strongylid eggs and Isospora sp. oocysts were found. Three monkeys from La Pacifica died and were examined for adult helminths. They were infected with Ascaris lumbricoides, C. biliophilus and T. minutus.

Integrated cytokine and metabolic analysis of pathological responses to parasite exposure in rodents

Parasitic infections cause a myriad of responses in their mammalian hosts, on immune as well as on metabolic level. A multiplex panel of cytokines and metabolites derived from four parasite-rodent models, namely, Plasmodium berghei-mouse, Trypanosoma brucei brucei-mouse, Schistosoma mansoni-mouse, and Fasciola hepatica-rat were statistically coanalyzed. 1H NMR spectroscopy and multivariate statistical analysis were used to characterize the urine and plasma metabolite profiles in infected and noninfected animals. Each parasite generated a unique metabolic signature in the host. Plasma cytokine concentrations were obtained using the ‘Meso Scale Discovery’ multi cytokine assay platform. Multivariate data integration methods were subsequently used to elucidate the component of the metabolic signature which is associated with inflammation and to determine specific metabolic correlates with parasite-induced changes in plasma cytokine levels. For example, the relative levels of acetyl glycoproteins extracted from the plasma metabolite profile in the P. berghei-infected mice were statistically correlated with IFN-γ, whereas the same cytokine was anticorrelated with glucose levels. Both the metabolic and the cytokine data showed a similar spatial distribution in principal component analysis scores plots constructed for the combined murine data, with samples from all infected animals clustering according to the parasite species and whereby the protozoan infections (P. berghei and T. b. brucei) grouped separately from the helminth infection (S. mansoni). For S. mansoni, the main infection-responsive cytokines were IL-4 and IL-5, which covaried with lactate, choline, and D-3-hydroxybutyrate. This study demonstrates that the inherently differential immune response to single and multicellular parasites not only manifests in the cytokine expression, but also consequently imprints on the metabolic signature, and calls for in-depth analysis to further explore direct links between immune features and biochemical pathways.

Comparação entre técnicas coproparasitológicas no diagnóstico de ovos de helmintos e oocistos de protozoários em cães.

In this study we evaluated the frequency of enteroparasites in pet dogs and their association with age, sex and breed, as well as the efficiency of the Willis-Mollay, Faust, Sedimentation and Direct exam methods. By these methods we processed 401 fecal samples. The samples were positive in the following percentages: Ancylostoma spp. (53.1%), Toxocara canis (20.7%), Cystoisospora ohioensis (15.7%), Trichuris vulpis (3.7%), Dipylidium caninum (2.5%) and Taenia spp. (1.0%). Toxocara canis (67.3%) and C. ohioensis (47.3%) showed higher positivity in the puppies. The Willis-Mollay technique was more efficient in the diagnosis of Ancylostoma spp. and T. canis eggs. The Direct method was the least efficient. It was found that the majority of the cases of D. caninum were diagnosed by the Sedimentation method (8=2.0%), while for T. vulpis Willis-Mollay (12=3.0%) and Sedimentation (13=3.2%) were more efficient. In view of these results, we can recommend the association of Willis-Mollay and Sedimentation methods for the diagnosis of gastrointestinal helminths. Due to the elevated occurrence of Ancylostoma spp. and T. canis, which are involved in zoonotic diseases, it becomes necessary to apply more efficient prophylaxis of canine intestinal parasitosis at the City of Araçatuba, state of São Paulo.

Leishmanicidal activity of an alkenylphenol from Piper malacophyllum is related to plasma membrane disruption

Leishmaniasis and Chagas disease are parasitic protozoan infections that affect the poorest population in the world, causing high mortality and morbidity. As a result of highly toxic and long-duration treatments, novel, safe and more efficacious drugs are essential. In this work, the methanol (MeOH) extract from the leaves of Piper malacophyllum (Piperaceae) was fractioned to afford one alkenylphenol, which was characterized as 4-[(3'E)-decenyl]phenol (gibbilimbol B) by spectroscopic methods. Anti-protozoan in vitro assays demonstrated for the first time that Leishmania (L.) infantum chagasi was susceptible to gibbilimbol B. with an in vitro EC50 of 23 mu g/mL against axenic promastigotes and an EC50 of 22 mu g/mL against intracellular amastigotes. Gibbilimbol B was also tested for anti-trypanosomal activity (Trypanosoma cruzi) and showed an EC50 value of 17 mu g/mL against trypomastigotes. To evaluate the cytotoxic parameters, this alkenylphenol was tested in vitro against NCTC cells, showing a CC50 of 59 mu g/mL and absent hemolytic activity at the highest concentration of 75 mu g/mL. Using the fluorescent probe SYTOX Green suggested that the alkenylphenol disrupted the Leishmania plasma membrane upon initial incubation. Further drug design studies aiming at derivatives could be a promising tool for the development of new therapeutic agents for leishmaniasis and Chagas disease. (C) 2012 Elsevier Inc. All rights reserved.

Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Background: Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe3O4 MNPs in order to provoke cell death remotely using TVMFs. Methods: Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO4 in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry. Results: MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Conclusion: As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.

Immunological Interactions between 2 Common Pathogens, Th1-Inducing Protozoan Toxoplasma gondii and the Th2-Inducing Helminth Fasciola hepatica

Background: The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-gamma, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel

Background: Infectious diarrhea can be caused by bacteria, viruses, or protozoan organisms, or a combination of these. The identification of co-infections in dogs is important to determine the prognosis and to plan strategies for their treatment and prophylaxis. Although many pathogens have been individually detected with real-time polymerase chain reaction (PCR), a comprehensive panel of agents that cause diarrhea in privately owned dogs has not yet been established. The objective of this study was to use a real-time PCR diarrhea panel to survey the frequencies of pathogens and co-infections in owned dogs attended in a veterinary hospital with and without diarrhea, as well the frequency in different countries. Feces samples were tested for canine distemper virus, canine coronavirus, canine parvovirus type 2 (CPV-2), Clostridium perfringens alpha toxin (CPA), Cryptosporidium spp., Giardia spp., and Salmonella spp. using molecular techniques.Results: In total, 104 diarrheic and 43 control dogs that were presented consecutively at a major private veterinary hospital were included in the study. Overall, 71/104 (68.3%) dogs with diarrhea were positive for at least one pathogen: a single infection in 39/71 dogs (54.9%) and co-infections in 32/71 dogs (45.1%), including 21/32 dogs (65.6%) with dual, 5/32 (15.6%) with triple, and 6/32 (18.8%) with quadruple infections. In the control group, 13/43 (30.2%) dogs were positive, all with single infections only. The most prevalent pathogens in the diarrheic dogs were CPA (40/104 dogs, 38.5%), CPV-2 (36/104 dogs, 34.6%), and Giardia spp. (14/104 dogs, 13.5%). CPV-2 was the most prevalent pathogen in the dual co-infections, associated with CPA, Cryptosporidium spp., or Giardia spp. No statistical difference (P = 0.8374) was observed in the duration of diarrhea or the number of deaths (P = 0.5722) in the presence or absence of single or co-infections.Conclusions: Diarrheic dogs showed a higher prevalence of pathogen infections than the controls. Whereas the healthy dogs had only single infections, about half the diarrheic dogs had co-infections. Therefore, multiple pathogens should be investigated in dogs presenting with diarrhea. The effects of multiple pathogens on the disease outcomes remain unclear because the rate of death and the duration of diarrhea did not seem to be affected by these factors.

Transplant-Associated and Blood Transfusion-Associated Tropical and Parasitic Infections

Blood transfusion and transplantation may represent efficient mechanisms of spreading infectious agents to naive populations. In the developed countries, as a consequence of globalization, several factors such as international commerce, tourism, and immigration have acted as important features for the emergence or reemergence of infectious diseases previously referred to as tropical. This article reviews the relevant bacterial, protozoan and viral infections that are more frequently associated with blood transfusion and/or solid organ or marrow transplantation and may affect susceptible populations worldwide.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

Besnoitia besnoiti, an apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may dramatically affect body condition, and, in males, lead to irreversible infertility. While identification of clinical cases and their histopathological confirmation is relatively simple to carry out, finding subclinical forms of infection is more difficult, thus a more sensitive test for the identification of the etiological agent may be an appropriate diagnostic tool. We have developed the ITS1 rDNA-sequence-based conventional and real-time PCR which are highly sensitive and specific for the detection of B. besnoiti infection in cattle. A recombinant internal positive control was introduced to assess possible sample-related inhibitory effects during the amplification reaction and, in order to prevent false-positive results, a pre-PCR treatment of potentially contaminating dU-containing PCR product with uracil-DNA-glycosylase (UDG) was followed.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Recent insights into the mucosal reactions associated with Giardia lamblia infections.

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.