Neospora caninum and bone marrow-derived dendritic cells: parasite survival, proliferation, and induction of cytokine expression

Dendritic cells (DCs) represent the first line defence of the innate immune system following infection with pathogens. We exploratively addressed invasion and survival ability of Neospora caninum, a parasite causing abortion in cattle, in mouse bone marrow DCs (BMDCs), and respective cytokine expression patterns. Immature BMDCs were exposed to viable (untreated) and nonviable parasites that had been inactivated by different means. Invasion and/or internalization, as well as intracellular survival and proliferation of tachyzoites were determined by NcGRA2-RT-PCR and transmission electron microscopy (TEM). Cytokine expression was evaluated by reverse transcription (RT)-PCR and cytokine ELISA. Transmission electron microscopy of DCs stimulated with untreated viable parasites revealed that N. caninum was able to invade and proliferate within BMDCs. This was confirmed by NcGRA2-RT-PCR. On the other hand, no viable parasite organisms were revealed by TEM when exposing BMDCs to inactivated parasites (nonviability demonstrated by NcGRA2-RT-PCR). Cytokine expression analysis (as assessed by both RT-PCR and ELISA) demonstrated that both viable and nonviable parasites stimulated mBMDCs to express IL-12p40, IL-10 and TNF-alpha, whereas IL-4 RNA expression was not detected. Thus, exposure of mBMDCs to both viable and nonviable parasites results in the expression of cytokines that are relevant for a mixed Th1/Th2 immune response.

Intermediate hosts of Toxoplasma gondii and food safety:epidemiology, genetics and parasite survival in meat-producing animals in Italy

Toxoplasma gondii is a coccidian parasite with a global distribution. The definitive host is the cat (and other felids). All warm-blooded animals can act as intermediate hosts, including humans. Sexual reproduction (gametogony) takes place in the final host and oocysts are released in the environment, where they then sporulate to become infective. In intermediate hosts the cycle is extra-intestinal and results in the formation of tachyzoites and bradyzoites. Tachyzoites represent the invasive and proliferative stage and on entering a cell it multiplies asexually by endodyogeny. Bradyzoites within tissue cysts are the latent form. T. gondii is a food-borne parasite causing toxoplasmosis, which can occur in both animals and humans. Infection in humans is asymptomatic in more than 80% of cases in Europe and North-America. In the remaining cases patients present fever, cervical lymphadenopathy and other non-specific clinical signs. Nevertheless, toxoplasmosis is life threatening if it occurs in immunocompromised subjects. The main organs involved are brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes, pancreas and parasite can be isolated from these tissues. Another aspect is congenital toxoplasmosis that may occur in pregnant women and the severity of the consequences depends on the stage of pregnancy when maternal infection occurs. Acute toxoplasmosis in developing foetuses may result in blindness, deformation, mental retardation or even death. The European Food Safety Authority (EFSA), in recent reports on zoonoses, highlighted that an increasing numbers of animals resulted infected with T. gondii in EU (reported by the European Member States for pigs, sheep, goats, hunted wild boar and hunted deer, in 2011 and 2012). In addition, high prevalence values have been detected in cats, cattle and dogs, as well as several other animal species, indicating the wide distribution of the parasite among different animal and wildlife species. The main route of transmission is consumption of food and water contaminated with sporulated oocysts. However, infection through the ingestion of meat contaminated with tissue cysts is frequent. Finally, although less frequent, other food products contaminated with tachyzoites such as milk, may also pose a risk. The importance of this parasite as a risk for human health was recently highlighted by EFSA’s opinion on modernization of meat inspection, where Toxoplasma gondii was identified as a relevant hazard to be addressed in revised meat inspection systems for pigs, sheep, goats, farmed wild boar and farmed deer (Call for proposals -GP/EFSA/BIOHAZ/2013/01). The risk of infection is more highly associated to animals reared outside, also in free-range or organic farms, where biohazard measure are less strict than in large scale, industrial farms. Here, animals are kept under strict biosecurity measures, including barriers, which inhibit access by cats, thus making soil contamination by oocysts nearly impossible. A growing demand by the consumer for organic products, coming from free-range livestock, in respect of animal-welfare, and the desire for the best quality of derived products, have all led to an increase in the farming of free-range animals. The risk of Toxoplasma gondii infection increases when animals have access to environment and the absence of data in Italy, together with need for in depth study of both the prevalence and genotypes of Toxoplasma gondii present in our country were the main reasons for the development of this thesis project. A total of 152 animals have been analyzed, including 21 free-range pigs (Suino Nero race), 24 transhumant Cornigliese sheep, 77 free-range chickens and 21 wild animals. Serology (on meat juice) and identification of T. gondii DNA through PCR was performed on all samples, except for wild animals (no serology). An in-vitro test was also applied with the aim to find an alternative and valid method to bioassay, actually the gold standard. Meat samples were digested and seeded onto Vero cells, checked every day and a RT-PCR protocol was used to determine an eventual increase in the amount of DNA, demonstrating the viability of the parasite. Several samples were alos genetically characterized using a PCR-RFLP protocol to define the major genotypes diffused in the geographical area studied. Within the context of a project promoted by Istituto Zooprofilattico of Pavia and Brescia (Italy), experimentally infected pigs were also analyzed. One of the aims was to verify if the production process of cured “Prosciutto di Parma” is able to kill the parasite. Our contribution included the digestion and seeding of homogenates on Vero cells and applying the Elisa test on meat juice. This thesis project has highlighted widespread diffusion of T. gondii in the geographical area taken into account. Pigs, sheep, chickens and wild animals showed high prevalence of infection. The data obtained with serology were 95.2%, 70.8%, 36.4%, respectively, indicating the spread of the parasite among numerous animal species. For wild animals, the average value of parasite infection determined through PCR was 44.8%. Meat juice serology appears to be a very useful, rapid and sensitive method for screening carcasses at slaughterhouse and for marketing “Toxo-free” meat. The results obtained on fresh pork meat (derived from experimentally infected pigs) before (on serum) and after (on meat juice) slaughter showed a good concordance. The free-range farming put in evidence a marked risk for meat-producing animals and as a consequence also for the consumer. Genotyping revealed the diffusion of Type-II and in a lower percentage of Type-III. In pigs is predominant the Type-II profile, while in wildlife is more diffused a Type-III and mixed profiles (mainly Type-II/III). The mixed genotypes (Type-II/III) could be explained by the presence of mixed infections. Free-range farming and the contact with wildlife could facilitate the spread of the parasite and the generation of new and atypical strains, with unknown consequences on human health. The curing process employed in this study appears to produce hams that do not pose a serious concern to human health and therefore could be marketed and consumed without significant health risk. Little is known about the diffusion and genotypes of T. gondii in wild animals; further studies on the way in which new and mixed genotypes may be introduced into the domestic cycle should be very interesting, also with the use of NGS techniques, more rapid and sensitive than PCR-RFLP. Furthermore wildlife can become a valuable indicator of environmental contamination with T. gondii oocysts. Other future perspectives regarding pigs include the expansion of the number of free-range animals and farms and for Cornigliese sheep the evaluation of other food products as raw milk and cheeses. It should be interesting to proceed with the validation of an ELISA test for infection in chickens, using both serum and meat juice on a larger number of animals and the same should be done also for wildlife (at the moment no ELISA tests are available and MAT is the reference method for them). Results related to Parma ham do not suggest a concerning risk for consumers. However, further studies are needed to complete the risk assessment and the analysis of other products cured using technological processes other than those investigated in the present study. For example, it could be interesting to analyze products such as salami, produced with pig meat all over the Italian country, with very different recipes, also in domestic and rural contexts, characterized by a very short period of curing (1 to 6 months). Toxoplasma gondii is one of the most diffuse food-borne parasites globally. Public health safety, improved animal production and protection of endangered livestock species are all important goals of research into reliable diagnostic tools for this infection. Future studies into the epidemiology, parasite survival and genotypes of T. gondii in meat producing animals should continue to be a research priority.

Anopheles gambiae eicosanoids modulate Plasmodium berghei survival from oocyst to salivary gland invasion

Eicosanoids affect the immunity of several pathogen/insect models, but their role on the Anopheles gambiae response to Plasmodium is still unknown. Plasmodium berghei-infected mosquitoes were injected with an eicosanoid biosynthesis inhibitor, indomethacin (IN), or a substrate, arachidonic acid (AA), at day 7 or day 12 post-infection (p.i.). Salivary gland invasion was evaluated by sporozoite counts at day 21 p.i. IN promoted infection upon sporozoite release from oocysts, but inhibited infection when sporozoites were still maturing within the oocysts, as observed by a reduction in the number of sporozoites reaching the salivary glands. AA treatment had the opposite effect. We show for the first time that An. gambiae can modulate parasite survival through eicosanoids by exerting an antagonistic or agonistic effect on the parasite, depending on its stage of development.

Host sex and ectoparasites choice: preference for, and higher survival on female hosts.

1. Sex differences in levels of parasite infection are a common rule in a wide range of mammals, with males usually more susceptible than females. Sex-specific exposure to parasites, e.g. mediated through distinct modes of social aggregation between and within genders, as well as negative relationships between androgen levels and immune defences are thought to play a major role in this pattern. 2. Reproductive female bats live in close association within clusters at maternity roosts, whereas nonbreeding females and males generally occupy solitary roosts. Bats represent therefore an ideal model to study the consequences of sex-specific social and spatial aggregation on parasites' infection strategies. 3. We first compared prevalence and parasite intensities in a host-parasite system comprising closely related species of ectoparasitic mites (Spinturnix spp.) and their hosts, five European bat species. We then compared the level of parasitism between juvenile males and females in mixed colonies of greater and lesser mouse-eared bats Myotis myotis and M. blythii. Prevalence was higher in adult females than in adult males stemming from colonial aggregations in all five studied species. Parasite intensity was significantly higher in females in three of the five species studied. No difference in prevalence and mite numbers was found between male and female juveniles in colonial roosts. 4. To assess whether observed sex-biased parasitism results from differences in host exposure only, or, alternatively, from an active, selected choice made by the parasite, we performed lab experiments on short-term preferences and long-term survival of parasites on male and female Myotis daubentoni. When confronted with adult males and females, parasites preferentially selected female hosts, whereas no choice differences were observed between adult females and subadult males. Finally, we found significantly higher parasite survival on adult females compared with adult males. 5. Our study shows that social and spatial aggregation favours sex-biased parasitism that could be a mere consequence of an active and adaptive parasite choice for the more profitable host.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that-at its mature stage-consists of an inner germinal and an outer laminated layer (GL ; LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host-parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta+CD4+T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8+T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels,

The intracellular parasite Theileria parva protects infected T cells from apoptosis.

Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells.

Caractérisation d’un effecteur de phosphoinositides chez le parasite de la malaria "Plasmodium falciparum"

La malaria est une maladie infectieuse causant plus de 500 000 morts chaque année. La maladie est causée par un protozoaire de la famille Plasmodium. L’apparition de souches résistantes aux traitements actuels et l’absence de vaccin efficace rendent la découverte de nouvelles cibles thérapeutiques urgente. Le parasite possède un complexe apical, un groupement de vacuoles sécrétoires spécialisées contenant les protéines responsables de l’invasion du globule rouge. Nous nous intéressons aux mécanismes gouvernant le transport intracellulaire de ces protéines et à la biogenèse du complexe apical lors de la formation des nouveaux parasites. Plus particulièrement, nous nous intéressons au rôle des phosphoinositides dans le recrutement des protéines à la membrane de l’appareil de Golgi. Par analyse bio-informatique du génome de P. falciparum, nous avons identifié plusieurs protéines effectrices liant potentiellement les phosphoinositides. Les travaux présentés dans ce mémoire concernent Mal13P1.188, une protéine possédant un domaine Pleckstrin homology. Nous proposons que Mal13P1.188 ait un rôle dans la génération du complexe apical en recrutant les protéines le constituant à la membrane du Golgi par la liaison avec les phosphoinositides. Afin de vérifier nos hypothèses, nous avons généré une lignée de parasite dont le gène de Mal13P1.188 est fusionné avec une GFP et une hémagglutinine. À l’aide de cette lignée de parasite, nous avons pu identifier Mal13P1.188 à proximité de l’appareil de Golgi lorsque les parasites étaient sous la forme schizont du cycle érythrocytaire. D’autres expériences ont permis de confirmer que le domaine Pleckstrin homology de Mal13P1.188 était capable de reconnaître les différentes formes de phosphoinositides. Finalement, d’autres travaux devront être faits sur Mal13P1.188 afin de déterminer si elle est essentielle à la survie du parasite.

BALB/c Mice Infected with Antimony Treatment Refractory Isolate of Leishmania braziliensis Present Severe Lesions due to IL-4 Production

Background: Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis-infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult. Methodology/Principal Findings: Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-gamma, TNF-alpha, IL-10 and TGF-beta were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S). Conclusion/Significance: We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC(50) = 0.89+/-0.02 mM at 28 degrees C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54+/-0.01 mM at 37 degrees C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress.

Hypoxia modulates phenotype, inflammatory response, and leishmanial infection of human dendritic cells

Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA-DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL-12p70, the bioactive interleukin-12 (IL-12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro-inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.

Secretory pathway of trypanosomatid parasites

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Tryanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles it? the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory), transport and protein glycosylation that may be exploited in developing new antiparasite drugs.

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

Interactions between Leishmania mexicana mexicana promastigotes and amastigotes and murine peritoneal macrophages in vitro

Unstimulated adherent mouse peritoneal cells were cultured in vitro and infected with equal numbers of a single strain of Leishmania m. mexicana amastigotes (AM), virulent promastigotes (VP), avirulent promastigotes (AVP) and fixed promastigotes (FP). Duplicate May-Grünwald-Giemsa stained coverslips were examined at time intervals up to 13 days. By 3 hr post infection, the number of macrophages containing parasites varied between 60.5% (VP) and 84% (AM) for macrophages exposed to living parasites, compared to 6.5% for macrophages exposed for FP. However, variable numbers of parasites showed degenerative changes by 3 hr, and the number of macrophages containing morphologically intact parasites varied significantly between cells infected with AM (84%) and those infected with VP (42%) or AVP(40%). The mean number on intacte parasites/macrophage also differed significantly between AM-infected cells and living or fixed promastigotes-infected cells. Quantitation of intact and degenerated parasites indicated parasite multiplication, as well as destruction, in VP-infected cells and parasite survival and multiplication in AM-infecte monolayers; in contrast no evidence of parasite multiplication was seen in AVP-infected cells. Changes in the mono layer itself (cell loss and macrophage vacuolization) were also evaluated. These results suggest that crucial events determining the outcome of infection occur in the host-parasite relationship during the fist 24 hours of infection. These events are apparently influenced not only by parasite or host strain but by environmentally induced variation within a given strain.