How many parasites species a frog might have? determinants of parasite diversity in South American anurans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

A research agenda for malaria eradication: basic science and enabling technologies

Today's malaria control efforts are limited by our incomplete understanding of the biology of Plasmodium and of the complex relationships between human populations and the multiple species of mosquito and parasite. Research priorities include the development of in vitro culture systems for the complete life cycle of P. falciparum and P. vivax and the development of an appropriate liver culture system to study hepatic stages. In addition, genetic technologies for the manipulation of Plasmodium need to be improved, the entire parasite metabolome needs to be characterized to identify new druggable targets, and improved information systems for monitoring the changes in epidemiology, pathology, and host-parasite-vector interactions as a result of intensified control need to be established to bridge the gap between bench, preclinical, clinical, and population-based sciences.

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,

Host and Environmental Influences on Development of Disease

While many myxozoan parasites produce asymptomatic infections in fish hosts, several species cause diseases whose patterns of prevalence and pathogenicity are highly dependent on host and environmental factors. This chapter reviews how these factors influence pathogenicity and disease prevalence. Influential host factors include age, size and nutritional state. There is also strong evidence for host strains that vary in resistance to infection and that there is a genetic basis for resistance. A lack of co-evolutionary processes appears to generally underly the devastating impacts of diseases caused by myxozoans when introduced fish are exposed to novel parasites (e.g. PKD in rainbow trout in Europe) or when native fish are exposed to an introduced parasite (e.g. whirling disease in North America). Most available information on abiotic factors relates to water temperature, which has been shown to play a crucial role in several host parasite systems (e.g. whirling disease, PKD) and is therefore of concern in view of global warming, fish health and food sustainability. Eutrophication may also influence disease development. Abiotic factors may also drive fish disease via their impact on parasite development in invertebrate hosts.

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development.

BACKGROUND The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. RESULTS Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. CONCLUSIONS Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.

Living on the edge: parasite taxonomy in Australia

The way in which the huge Australian parasite fauna is described (discovery and naming) is the subject of this address. The approach to the task has never been well-organised so that a few groups of parasites are now relatively well-known because of the efforts of small groups of workers who have made sustained efforts in these groups, but equally some host-parasite systems have been almost completely ignored in that no worker has ever given them sustained attention. A high proportion of Australian parasites have been described by international workers; The sustaining of interest in a group of parasites over a long period is the key to real progress being made. The nature of the organisation of Australian science presently means that few positions are available for parasite taxonomists and funding for taxonomic research is scarce. Thus, parasite taxonomy (like the taxonomy of many groups of Australian plants and animals) can only be considered to be in crisis. (C) 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Exposing extinction risk analysis to pathogens: Is disease just another form of density dependence?

In the United States and several other countries., the development of population viability analyses (PVA) is a legal requirement of any species survival plan developed for threatened and endangered species. Despite the importance of pathogens in natural populations, little attention has been given to host-pathogen dynamics in PVA. To study the effect of infectious pathogens on extinction risk estimates generated from PVA, we review and synthesize the relevance of host-pathogen dynamics in analyses of extinction risk. We then develop a stochastic, density-dependent host-parasite model to investigate the effects of disease on the persistence of endangered populations. We show that this model converges on a Ricker model of density dependence under a suite of limiting assumptions, including. a high probability that epidemics will arrive and occur. Using this modeling framework, we then quantify: (1) dynamic differences between time series generated by disease and Ricker processes with the same parameters; (2) observed probabilities of quasi-extinction for populations exposed to disease or self-limitation; and (3) bias in probabilities of quasi-extinction estimated by density-independent PVAs when populations experience either form of density dependence. Our results suggest two generalities about the relationships among disease, PVA, and the management of endangered species. First, disease more strongly increases variability in host abundance and, thus, the probability of quasi-extinction, than does self-limitation. This result stems from the fact that the effects and the probability of occurrence of disease are both density dependent. Second, estimates of quasi-extinction are more often overly optimistic for populations experiencing disease than for those subject to self-limitation. Thus, although the results of density-independent PVAs may be relatively robust to some particular assumptions about density dependence, they are less robust when endangered populations are known to be susceptible to disease. If potential management actions involve manipulating pathogens, then it may be useful to. model disease explicitly.

Apicomplexans pulling the strings: manipulation of the host cell cytoskeleton dynamics

Invasive stages of apicomplexan parasites require a host cell to survive, proliferate and advance to the next life cycle stage. Once invasion is achieved, apicomplexans interact closely with the host cell cytoskeleton, but in many cases the different species have evolved distinct mechanisms and pathways to modulate the structural organization of cytoskeletal filaments. The host cell cytoskeleton is a complex network, largely, but not exclusively, composed of microtubules, actin microfilaments and intermediate filaments, all of which are modulated by associated proteins, and it is involved in diverse functions including maintenance of cell morphology and mechanical support, migration, signal transduction, nutrient uptake, membrane and organelle trafficking and cell division. The ability of apicomplexans to modulate the cytoskeleton to their own advantage is clearly beneficial. We here review different aspects of the interactions of apicomplexans with the three main cytoskeletal filament types, provide information on the currently known parasite effector proteins and respective host cell targets involved, and how these interactions modulate the host cell physiology. Some of these findings could provide novel targets that could be exploited for the development of preventive and/or therapeutic strategies.

Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?

Spatial disease ecology is emerging as a new field that requires the integration of complementary approaches to address how the distribution and movements of hosts and parasites may condition the dynamics of their interactions. In this context, migration, the seasonal movement of animals to different zones of their distribution, is assumed to play a key role in the broad scale circulation of parasites and pathogens. Nevertheless, migration is not the only type of host movement that can influence the spatial ecology, evolution, and epidemiology of infectious diseases. Dispersal, the movement of individuals between the location where they were born or bred to a location where they breed, has attracted attention as another important type of movement for the spatial dynamics of infectious diseases. Host dispersal has notably been identified as a key factor for the evolution of host-parasite interactions as it implies gene flow among local host populations and thus can alter patterns of coevolution with infectious agents across spatial scales. However, not all movements between host populations lead to dispersal per se. One type of host movement that has been neglected, but that may also play a role in parasite spread is prospecting, i.e., movements targeted at selecting and securing new habitat for future breeding. Prospecting movements, which have been studied in detail in certain social species, could result in the dispersal of infectious agents among different host populations without necessarily involving host dispersal. In this article, we outline how these various types of host movements might influence the circulation of infectious disease agents and discuss methodological approaches that could be used to assess their importance. We specifically focus on examples from work on colonial seabirds, ticks, and tick-borne infectious agents. These are convenient biological models because they are strongly spatially structured and involve relatively simple communities of interacting species. Overall, this review emphasizes that explicit consideration of the behavioral and population ecology of hosts and parasites is required to disentangle the relative roles of different types of movement for the spread of infectious diseases.

Protostrongylids nematodes as a tool to study the biogeography of wild mammals

The European brown hare (Lepus europaeus Pallas, 1778) is an important game species, distributed across Europe and introduced in other regions. Recently, a geographically isolated population, closely related to an ancestral lineage of Lepus europaeus meridiei, was found on Pianosa Island, off the coast of Tuscany, Italy (Mengoni et al., 2018). Thus, the unique opportunity to explore the evolution and genetic structure of its helminth parasites was added to its exceptional isolation condition. Various lungworm species within the genus Protostrongylus (Nematoda: Protostrongylidae) are described in European brown hares. Our aim was to analyze the parasite population through morphological and molecular approaches in order to study the biogeography of the European brown hares (L. e. meridiei) population from Pianosa Island. Moreover, we investigated the morphology of a monospecific genus, i.e. Orthostrongylus, considering its quite intrigant descriptive history and its still unclear and debated classification. Nuclear and mitochondrial markers were used based on their resolution power and expected polymorphism; the whole Internal Transcribed Spacer 1 and 2 (ITS), including the 5.8S rRNA sequence and the Large Subunit (28S) were used, as nuclear genes, for confirmation of the species identification. Conversely, the cytochrome oxidase c subunit I (COI) was used, as mithocondrial genes, to assess interspecific genetic relationships. Molecular analysis corroborated the morphological identification since all the generated ITS and LSU sequences were 100% consistent with the species Protostrongylus oryctolagi and Orthostrongylus macrotis. The paucity of molecular data existent about this genus of parasites underlines the need for more insight’s studies. An in-depth analysis of broncho-pulmonary parasites and the host-parasites relationships along with the improvement of the use of mitochondrial genes, as well as the assessment of new polymorphic markers could contribute to an extensive understanding of parasites fauna and taxonomy, as well as their relationship with wild mammals’ hosts.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

Non-coding RNAs in schistosomes: an unexplored world

Non-coding RNAs (ncRNAs) were recently given much higher attention due to technical advances in sequencing which expanded the characterization of transcriptomes in different organisms. ncRNAs have different lengths (22 nt to >1, 000 nt) and mechanisms of action that essentially comprise a sophisticated gene expression regulation network. Recent publication of schistosome genomes and transcriptomes has increased the description and characterization of a large number of parasite genes. Here we review the number of predicted genes and the coverage of genomic bases in face of the public ESTs dataset available, including a critical appraisal of the evidence and characterization of ncRNAs in schistosomes. We show expression data for ncRNAs in Schistosoma mansoni. We analyze three different microarray experiment datasets: (1) adult worms' large-scale expression measurements; (2) differentially expressed S. mansoni genes regulated by a human cytokine (TNF-α) in a parasite culture; and (3) a stage-specific expression of ncRNAs. All these data point to ncRNAs involved in different biological processes and physiological responses that suggest functionality of these new players in the parasite's biology. Exploring this world is a challenge for the scientists under a new molecular perspective of host-parasite interactions and parasite development.