Testing Local Adaptation in a Natural Great Tit-Malaria System: An Experimental Approach.

Finding out whether Plasmodium spp. are coevolving with their vertebrate hosts is of both theoretical and applied interest and can influence our understanding of the effects and dynamics of malaria infection. In this study, we tested for local adaptation as a signature of coevolution between malaria blood parasites, Plasmodium spp. and its host, the great tit, Parus major. We conducted a reciprocal transplant experiment of birds in the field, where we exposed birds from two populations to Plasmodium parasites. This experimental set-up also provided a unique opportunity to study the natural history of malaria infection in the wild and to assess the effects of primary malaria infection on juvenile birds. We present three main findings: i) there was no support for local adaptation; ii) there was a male-biased infection rate; iii) infection occurred towards the end of the summer and differed between sites. There were also site-specific effects of malaria infection on the hosts. Taken together, we present one of the few experimental studies of parasite-host local adaptation in a natural malaria system, and our results shed light on the effects of avian malaria infection in the wild.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp.)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in albumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern Brazil, compromising the weight of infected animals with clinical alterations appearing in severe cases of this disease. It was also noted that, although the hematological abnormalities presented by these animals may not have been conclusive, leukopenia, monocytosis and the decrease of basophils and lymphocytes revealed an unfavorable prognosis, and Plasmodium spp. infections may progress with elevated uric acid concentration and low albumin levels.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 µM melatonin and 0.1 µM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

Effects of autacoid inhibitors and of an antagonist on malaria infection in mice

The effects of p-chlorophenylalanine, an inhibitor of serotonin synthesis, indomethacin, an inhibitor of prostaglandin synthesis, cyproheptadine, a serotonin, bradykinin and histamine antagonist, were assessed separately and in combination with chloroquine (CQ) in Vom strains of Swiss albino mice (18-22 g) of either sex infected intraperitoneally with 1 x 10(7) Plasmodium yoelii nigeriensis-induced malaria. As prophylactic, these agents reduced from 31.9 ± 4.5 to 16.1 ± 8.1% the level of parasitemia relative to control but had no appreciable activity as curative agents when administered subcutaneously once daily for 4 days after 72 h of parasites innoculum in vivo. However, CQ alone and the combination of these agents with CQ in curative and prophylactic treatments significantly reduced (from 50.3 ± 5.8 to 4.9 ± 0.75%) the level of parasitemia (P < 0.05), which was taken only once 72 h after the parasites innoculum. The prophylactic result was shown to produce better results than the curative treatment. The data indicate that inhibitors and an antagonist can reduce the parasitemia load (the extent of damage and the severity of infection) as well as enhance the effects of CQ when combined with it for malaria therapy. The study reveals that the production of autacoids in established infection renders autacoid inhibitors and an antagonist ineffective for radical cure in malarial mice; however, selective inhibition of local hormones implicated in the pathological manifestations of malaria infection by autacoid inhibitors and an antagonist may be a possible pathway to reduce the severity of infection and the associated tissue damage and to enhance the efficacy of available anti-malarials.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed more than 5000 parasites/µL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

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.

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites

Background: Severe malarial anaemia is a major complication of malaria infection and is multifactorial resulting from loss of circulating red blood cells (RBCs) from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. Methods: Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. Results: Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells). Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. Conclusion: This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.

Gradual Decline in Malaria-Specific Memory T Cell Responses Leads to Failure to Maintain Long-Term Protective Immunity to Plasmodium chabaudi AS Despite Persistence of B Cell Memory and Circulating Antibody

The mechanisms responsible for the generation and maintenance of immunological memory to Plasmodium are poorly understood and the reasons why protective immunity in humans is so difficult to achieve and rapidly lost remain a matter for debate. A possible explanation for the difficulty in building up an efficient immune response against this parasite is the massive T cell apoptosis resulting from exposure to high-dose parasite Ag. To determine the immunological mechanisms required for long-term protection against P. chabaudi malaria and the consequences of high and low acute phase parasite loads for acquisition of protective immunity, we performed a detailed analysis of T and B cell compartments over a period of 200 days following untreated and drug-treated infections in female C57BL/6 mice. By comparing several immunological parameters with the capacity to control a secondary parasite challenge, we concluded that loss of full protective immunity is not determined by acute phase parasite load nor by serum levels of specific IgG2a and IgG1. Abs, but appears to be a consequence of the progressive decline in memory T cell response to parasites, which occurs similarly in untreated and drug-treated mice with time after infection. Furthermore, by analyzing adoptive transfer experiments, we confirmed the major role of CD4(+) T cells for guaranteeing long-term full protection against P. chabaudi malaria. The Journal of Immunology, 2008, 181: 8344-8355.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)

The circumsporozoite protein (CSP) of Plasmodium vivax, a major target for malaria vaccine development, has immunodominant B-cell epitopes mapped to central nonapeptide repeat arrays. To determine whether rearrangements of repeat motifs during mitotic DNA replication of parasites create significant CSP diversity under conditions of low effective meiotic recombination rates, we examined csp alleles from sympatric P. vivax isolates systematically sampled from an area of low malaria endemicity in Brazil over a period of 14 months. Nine unique csp types, comprising six different nona peptide repeats, were observed in 45 isolates analyzed. Identical or nearly identical repeats predominated in most arrays, consistent with their recent expansion. We found strong linkage disequilibrium at sites across the chromosome 8 segment flanking the csp locus, consistent with rare meiotic recombination in this region. We conclude that CSP repeat diversity may not be severely constrained by rare meiotic recombination in areas of low malaria endemicity. New repeat variants may be readily created by nonhomologous recombination even when meiotic recombination is rare, with potential implications for CSP-based vaccine development. (C) 2010 Elsevier B.V. All rights reserved.

Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax

The population structure of Plasmodium vivax remains elusive. The markers of choice for large-scale population genetic studies of eukaryotes, short tandem repeats known as microsatellites, have been recently reported to be less polymorphic in R vivax. Here we investigate the microsatellite diversity and geographic structure in P vivax, at both local and global levels, using 14 new markers consisting of tri- or tetranucleotide repeats. The local-level analysis, which involved 50 field isolates from Sri Lanka, revealed unexpectedly high diversity (average virtual heterozygosity [H-E], 0.807) and significant multilocus linkage disequilibrium in this region of low malaria endemicity. Multiple-clone infections occurred in 60% of isolates sampled in 2005. The global-level analysis of field isolates or monkey-adapted strains identified 150 unique haplotypes among 164 parasites from four continents. Individual P. vivax isolates could not be unambiguously assigned to geographic populations. For example, we found relatively low divergence among parasites from Central America, Africa, Southeast Asia and Oceania, but substantial differentiation between parasites from the same continent (South Asia and Southeast Asia) or even from the same country (Brazil). Parasite relapses, which may extend the duration of P. vivax carriage in humans, are suggested to facilitate the spread of strains across continents, breaking down any pre-existing geographic structure. (C) 2008 Elsevier B.V. All rights reserved.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 angstrom and T. cruzi at 1.9 angstrom resolution, and that from P. falciparum at a higher resolution (2.0 angstrom) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.