A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

Transmission blocking vaccines to control insect-borne diseases: a review

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.

Life cycle of Triatoma ryckmani (Hemiptera: Reduviidae) in the laboratory, feeding patterns in nature and experimental infection with Trypanosoma cruzi

A cohort initiated with 121 eggs, yielding 105 first instar nymphs (eclosion rate: 86.78%), allowed us to observe the entire life cycle of Triatoma ryckmani under laboratory conditions (24ºC and 62% relative humidity), by feeding them on anesthetized hamsters. It was possible to obtain 62 adults and the cycle from egg to adult took a mean of 359.69 days with a range of 176-529 days (mortality rate of nymphs: 40.95%). Mean life span of adults was of 81 days for females and 148 days for males. The developmental periods of 4th and 5th nymphs were longer than those of the other instars. This suggests that young siblings have a better chance of taking a hemolymph meal from older ones, in order to survive during fasting periods during prolonged absences of vertebrate hosts from natural ecotopes. The stomach contents of 37 insects showed blood from rodents (15 cases), lizards (7 cases), birds (6 cases) and insect hemolymph (7 cases). Out of 10 insects fed by xenodiagnosis on a Trypanosoma cruzi infected mouse, all but one became infected with the parasite.

Omental and pleural milky spots: different reactivity patterns in mice infected with Schistosoma mansoni reveals coelomic compartmentalisation

In vertebrate animals, pleural and peritoneal cavities are repositories of milky spots (MS), which constitute an organised coelom-associated lymphomyeloid tissue that is intensively activated by Schistosoma mansoni infection. This study compared the reactive patterns of peritoneal MS to pleural MS and concluded from histological analysis that they represent independent responsive compartments. Whole omentum, lungs and the entire mediastinum of 54 S. mansoni-infected mice were studied morphologically. The omental MS of infected animals were highly activated, modulating from myeloid-lymphocytic (60 days of infection) to lymphomyeloid (90 days of infection) and lymphocytic or lymphoplasmacytic (160 days of infection) types. The non-lymphoid component predominated in the acute phase of infection and was expressed by monocytopoietic, eosinopoietic and neutropoietic foci, with isolated megakaryocytes and small foci of late normoblasts and mast cells. Nevertheless, pleural or thoracic MS of infected mice were monotonous, consisting of small and medium lymphocytes with few mast and plasma cells and no myeloid component. Our data indicate that compartmentalisation of the MS response is dependent on the lymphatic vascularisation of each coelomic cavity, limiting the effects or consequences of any stimulating or aggressive agents, as is the case with S. mansoni infection.

Characterization of TcSTI-1, a homologue of stress-induced protein-1, in Trypanosoma cruzi

The life cycle of the protozoan Trypanosoma cruzi exposes it to several environmental stresses in its invertebrate and vertebrate hosts. Stress conditions are involved in parasite differentiation, but little is known about the stress response proteins involved. We report here the first characterization of stress-induced protein-1 (STI-1) in T. cruzi (TcSTI-1). This co-chaperone is produced in response to stress and mediates the formation of a complex between the stress proteins HSP70 and HSP90 in other organisms. Despite the similarity of TcSTI-1 to STI-1 proteins in other organisms, its expression profile in response to various stress conditions, such as heat shock, acidic pH or nutrient starvation, is quite different. Neither polysomal mRNA nor protein levels changed in exponentially growing epimastigotes cultured under any of the stress conditions studied. Increased levels of TcSTI-1 were observed in epimastigotes subjected to nutritional stress in the late growth phase. Co-immunoprecipitation assays revealed an association between TcSTI-1 and TcHSP70 in T. cruzi epimastigotes. Immunolocalization demonstrated that TcSTI-1 was distributed throughout the cytoplasm and there was some colocalization of TcSTI-1 and TcHSP70 around the nucleus. Thus, TcSTI-1 associates with TcHSP70 and TcSTI-1 expression is induced when the parasites are subjected to stress conditions during specific growth phase.

Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

The significance of the amoebocyte-producing organ in Biomphalaria glabrata

In molluscs, internal defence against microorganisms is performed by a single cell type, i.e., the haemocyte or amoebocyte. The origin of these cells in Biomphalaria glabrata was initially thought to be localised within the vasculo-connective tissue. More recently, origin from a single organ, termed the amoebocyte-producing organ (APO), has been postulated based on the occurrence of hyperplasia and mitoses during Schistosoma mansoni infection. The present investigation represents a histological, immuno-histochemical and ultra-structural study of the B. glabrata APO, whereby histological identification was facilitated by means of collecting epithelial basophilic cells. These cells were comprised of single-cell layers that cover a portion of the stroma, which contains many small, round cells and haemolymph sinuses, as well as a small area of the pericardial surface of the reno-pericardial region. On occasion, this epithelial component vaguely resembled the vertebrate juxtaglomerular apparatus, which reinforces its presumed relationship to the kidney. Both in normal and infected molluscs, mitoses were only occasionally found. The present quantitative studies failed to demonstrate the presence of APO cellular hyperplasia, either in normal or schistosome-infected B. glabrata. Conversely, several structural details from the APO region in B. glabrata were found to be consistent with the hypothesis that the APO is a filtration organ, i.e., it is more closely related to the kidney rather than the bone marrow, as has been suggested in the literature.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

A Microtus fortisprotein, serum albumin, is a novel inhibitor of Schistosoma japonicumschistosomula

Schistosomiasis is an endemic parasite disease and praziquantel is the only drug currently in use to control this disease. Experimental and epidemiological evidence strongly suggests that Microtus fortis( Mf) is a naturally resistant vertebrate host of Schistosoma japonicum. In the present study, we found that Mfserum albumin ( Mf-albumin) and the conditioned medium of pcDNA3.1- Mf-albumin caused 46.2% and 38.7% schistosomula death rates in 96 h, respectively, which were significantly higher than that of the negative control (p < 0.05). We also found that mice injected with Mf-albumin had a 43.5% reduction in worm burden and a 48.1% reduction in liver eggs per gram (p < 0.05) in comparison to the control animals. To characterise the mechanisms involved in clearance, schistosomula were incubated with fluorescein isothiocyanate-labelled Mf-albumin and fluorescent enrichment effects were found in the gut lumen of schistosomula after 48 h of incubation. Next, digestive tract excretions from schistosomula were collected and the sensitivity of Mf-albumin to digestive tract excretions was evaluated. The results indicated that schistosomula digestive tract excretions showed indigestibility of Mf-albumin. The death of schistosomula could be partially attributed to the lack of digestion of Mf-albumin by digestive tract excretions during the development of the schistosomula stage. Therefore, these data indicate the potential of Mf-albumin as one of the major selective forces for schistosomiasis.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.

Behavioural biology of Chagas disease vectors

Many arthropod species have adopted vertebrate blood as their main food source. Blood is rich in nutrients and, except for the presence of parasites, sterile. However, this food source is not freely available, nor is obtaining it devoid of risk. It circulates inside vessels hidden underneath the skin of mobile hosts that are able to defend themselves and even predate the insects that try to feed on them. Thus, the haematophagous lifestyle is associated with major morphological, physiological and behavioural adaptations that have accumulated throughout the evolutionary history of the various lineages of blood-sucking arthropods. These adaptations have significant consequences for the evolution of parasites as well as for the epidemiology of vector-transmitted diseases. In this review article, we analyse various aspects of the behaviour of triatomine bugs to illustrate how each behavioural trait represents a particular adaptation to their close association with their hosts, which may easily turn into predators. Our aim is to offer to the reader an up-to-date integrative perspective on the behaviour of Chagas disease vectors and to propose new research avenues to encourage both young and experienced colleagues to explore this aspect of triatomine biology.

Circadian rhythms in insect disease vectors

Organisms from bacteria to humans have evolved under predictable daily environmental cycles owing to the Earth’s rotation. This strong selection pressure has generated endogenous circadian clocks that regulate many aspects of behaviour, physiology and metabolism, anticipating and synchronising internal time-keeping to changes in the cyclical environment. In haematophagous insect vectors the circadian clock coordinates feeding activity, which is important for the dynamics of pathogen transmission. We have recently witnessed a substantial advance in molecular studies of circadian clocks in insect vector species that has consolidated behavioural data collected over many years, which provided insights into the regulation of the clock in the wild. Next generation sequencing technologies will facilitate the study of vector genomes/transcriptomes both among and within species and illuminate some of the species-specific patterns of adaptive circadian phenotypes that are observed in the field and in the laboratory. In this review we will explore these recent findings and attempt to identify potential areas for further investigation.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus.Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts inR. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.