Trematode genetic patterns at host individual and population scales provide insights about infection mechanisms.

Multiple parasites can infect a single host, creating a dynamic environment where each parasite must compete over host resources. Such interactions can cause greater harm to the host than single infections and can also have negative consequences for the parasites themselves. In their first intermediate hosts, trematodes multiply asexually and can eventually reach up to 20% of the host's biomass. In most species, it is unclear whether this biomass results from a single infection or co-infection by 2 or more infective stages (miracidia), the latter being more likely a priori in areas where prevalence of infection is high. Using as model system the trematode Bucephalus minimus and its first intermediate host cockles, we examined the genetic diversity of the cytochrome c oxidase subunit I region in B. minimus from 3 distinct geographical areas and performed a phylogeographic study of B. minimus populations along the Northeast Atlantic coast. Within localities, the high genetic variability found across trematodes infecting different individual cockles, compared to the absence of variability within the same host, suggests that infections could be generally originating from a single miracidium. On a large spatial scale, we uncovered significant population structure of B. minimus, specifically between the north and south of Bay of Biscay. Although other explanations are possible, we suggest this pattern may be driven by the population structure of the final host.

Effect of the alveolate parasite Perkinsus olseni infection on sexual maturation and spawning efficiency of the clam Ruditapes decussatus.

The effect of Perkinsus olseni infection on the reproduction ability of clams has been underestimated so far. Although some studies found evidence of reduction of egg production and delay in gonad maturation after infection, the total effect of the infection is still unclear. In this study, Ruditapes decussatus clams from a naïve population were injected with two different doses of P. olseni parasites, a low dose leading to a light infection and a high dose leading to a heavy infection. Clams were maintained during 2 months for maturation, and at the end of the experiment, the spawning was induced, the number of larvae release and mortality were evaluated. During the maturation period, infection level, gonadal stage, condition index, gross biochemical composition and oxidative status of progenitors were evaluated at days 0, 30 and 60 post-injection. The effects of P. olseni infection on clams showed alterations on biochemical parameters, namely lipid peroxidation, a significant mortality and a delayed gonad maturation, with a greater effect in the highly infected individuals. The reproductive capacity of the clams was impaired in both infected groups showing a lower production and a higher mortality rate of larvae. Finally, this study indicates that the production of natural beds with a high prevalence of P. olseni could be compromised by a deregulation of the natural reproduction cycle and a decrease in larvae production by infected animals, probably due to a combination of lower egg production and lower lipid reserves in larvae from infected clams.

Immunopathology and modulation induced by hookworms: From understanding to intervention.

Hookworm infection is considered the most prevalent human soil-transmitted helminth infection affecting approximately 500 million people and accounting for 3.2 million disability-adjusted life years lost annually. As with many other neglected tropical diseases, no international surveillance mechanisms that show accurate data on the prevalence of hookworm infection are in place, thus hindering strategies to control parasite transmission. In this review, we unravel the current knowledge in immunopathology and immunoregulation of hookworm infection and present discoveries in drug therapies based on the capability of hookworms to regulate inflammation to treat allergic, inflammatory and metabolic diseases. Additionally, we highlight potential vaccine development and treatments and propose avenues for further inquiry.

Immunological underpinnings of Ascaris infection, reinfection and co-infection and their associated co-morbidities.

Human ascariasis is the most common and prevalent neglected tropical disease and is estimated that ~819 million people are infected around the globe, accounting for 0.861 million years of disability-adjusted life years in 2017. Even with the existence of highly effective drugs, the constant presence of infective parasite eggs in the environment contribute to a high reinfection rate after treatment. Due to its high prevalence and broad geographic distribution Ascaris infection is associated with a variety of co-morbidities and co-infections. Here, we provide data from both experimental models and humans studies that illustrate how complex is the interaction of Ascaris with the host immune system, especially, in the context of reinfections, co-infections and associated co-morbidities.

Effect of light on the trematode Himasthla elongata: from cercarial behaviour to infection success.

The cockle Cerastoderma edule, a socioeconomically important bivalve of the northeast Atlantic, is host to several trematodes, including Himasthla elongata. In the life cycle of this trematode, cercariae (free-living stages) emerge from the first intermediate host, a snail, to infect cockles as second intermediate hosts. During their lifespan (less than 2 d), cercariae must ensure successful host-to-host transmission via the surrounding water and therefore are exposed to and impacted by different environmental conditions, including abiotic factors. Given that the light:dark cycle is one of the major drivers of behaviour in aquatic habitats, we aimed to determine the influence of light on cercariae and host behaviour based on 3 hypotheses. First, by having a benthic second intermediate host, these cercariae will display a photonegative orientation; second, and conversely, host behaviour will not be influenced by light; and third, cercariae infection success will be light-dependent. Results showed that cercariae display a photopositive orientation (first hypothesis rejected), displaying movements towards light. Host activity (evaluated by oxygen consumption) was similar among conditions, i.e. dark vs. light (second hypothesis accepted), but hosts acquired more parasites when experimentally infected in the dark (third hypothesis accepted). This light-dependent infection of the host is explained by a change of cercarial behaviour when exposed to light, decreasing their infection success. This study highlights that trematode responses to external conditions may be linked to successful life cycle completion rather than being altered by the host habitat. Light influence on cercarial behaviour resulted in increased infection success that may affect trematode population dynamics and their distributional range.

Monorchis parvus and Gymnophallus choledochus: two trematode species infecting cockles as first and second intermediate host.

The most deleterious stage of a trematode life cycle occurs in the first intermediate host where the parasite penetrates as a miracidium and asexually multiplicates in sporocysts or rediae. When infection advances, other organs can be occupied with severe effects on host individual health and population dynamics. Existing studies focused on these host/parasite systems are still scarce due to the usual low prevalence in ecosystems. Using cockles (Cerastoderma spp.) and two trematode species (Monorchis parvus and Gymnophallus choledochus) infecting these bivalves as first and second intermediate host, the present work aimed to (1) summarize the most relevant literature and (2) provide new information regarding this host/parasite system, taking advantage of a 21-year monthly database from Banc d'Arguin (France). This long-term monitoring showed that different trematode species display varying host size range preference (6-38 and 31-36 mm for M. parvus and G. choledochus, respectively). The occurrence of coinfection was lower than expected, raising some questions related to parasite interspecific competition. This review improved our understanding of the processes shaping the prevalence and distribution of parasitism. This study highlighted that beyond constant trematode assemblage monitoring, there is a need to identify the main predictors of rediae/sporocysts infection, such as the definitive host dynamics and miracidium infection processes, for future better management of host severe disease and mortality episodes.

How costly are metacercarial infections in a bivalve host? Effects of two trematode species on biochemical performance of cockles.

Bivalve stocks have been decreasing in the last decades largely due to emergent diseases and consequent mass mortality episodes. Cerastoderma edule (the edible cockle) is one of the most exploited bivalves in Europe and is among the most common hosts for trematodes, the most prevalent macroparasites in coastal waters but yet poorly studied. Therefore, in the present study, this bivalve species was used as host model to determine if trematode infection exerts a negative effect on bivalve energy metabolism and balance and if the tissues targeted by different trematodes influence the metabolic cost, with physiological and biochemical consequences. Cockles were experimentally infected with two trematode species, Himasthla elongata and Renicola roscovitus, that infect the foot and palps, respectively. Trematode infection exerted a negative effect on the metabolism of C. edule, the second intermediate host, by reduction of oxygen consumption. A different host biochemical response was found depending on trematode species, especially in regard to the level of oxygen consumption decrease and the preferential accumulation of lipids and glycogen. This study represents a step towards the understanding of host-trematode relationships that can be used to better predict potential conservation threats to bivalve populations and to maximize the success of stock and disease management.

Leishmania infantum induces expression of the negative regulatory checkpoint, CTLA-4, by human naïve CD8+ T cells.

AIMS: CD8+ T cells are important in mediating protective responses to intracellular pathogens. However, an uncontrolled response may lead to pathology. The role of CD8+ T cells in different clinical manifestations of human leishmaniasis is controversial and poorly understood. We aim to study the response of CD8+ T cells to the first exposure to different strains of Leishmania, seeking to correlate these findings with clinical manifestations of disease. METHODS AND RESULTS: We have evaluated the expression of granzyme A, inflammatory and anti-inflammatory cytokines, as well as CTLA-4 by human naïve CD8+ T cells exposed to Leishmania braziliensis and two different strains of Leishmania infantum in vitro. We observed that while exposure to L braziliensis induced an inflammatory profile, as measured by the expression of granzyme A, IFN-gamma and IL-17, as well as a higher IFN/IL-10 ratio, exposure to L infantum led to a regulatory profile, as measured by lower IFN/IL-10 ratio and higher expression of CTLA-4. CONCLUSION: These results may help explain why patients with the visceral clinical form present a weaker cellular response and, consequently, a worse outcome of the disease. The use of CTLA-4 checkpoint inhibitors may emerge as a potential immunotherapy to ameliorate the immune response in visceral leishmaniasis patients.

Co-infection with distinct Trypanosoma cruzi strains induces an activated immune response in human monocytes.

AIMS: The aim of the study was to evaluate the immune response triggered by the first contact of human monocytes with two T cruzi strains from distinct discrete typing units (DTUs) IV and V, and whether co-infection with these strains leads to changes in monocyte immune profiles, which could in turn influence the subsequent infection outcome. METHODS AND RESULTS: We evaluated the influence of in vitro single- and co-infection with AM64 and 3253 strains on immunological characteristics of human monocytes. Single infection of monocytes with AM64 or 3253 induced opposing anti-inflammatory and inflammatory responses, respectively. Co-infection was observed in over 50% of monocytes after 15 hours of culture, but this percentage dropped ten-fold after 72 hours. Co-infection led to high monocyte activation and an increased percentage of both IL-10 and TNF. The decreased percentage of co-infected cells observed after 72 hours was associated with a decreased frequency of TNF-expressing cells. CONCLUSION: Our results show that the exacerbated response observed in co-infection with immune-polarizing strains is associated with a decreased frequency of co-infected cells, suggesting that the activated response favours parasite control. These findings may have implications for designing new Chagas disease preventive strategies.

Spatio-temporal variation of trematode parasites community in Cerastoderma edule cockles from Ria de Aveiro (Portugal).

Cerastoderma edule (edible cockle) is among the most exploited bivalves in Europe playing an important socio-economic role. Cockles live in estuaries and lagoons where their population is controlled by several environmental factors including parasitism. Parasites represent an important part of the world known biodiversity but are often neglected. Trematodes are the most prevalent macroparasites of cockles being able to exert an impact both at the individual and population levels. Therefore, it is of prime relevance to recognize and understand the parasite-host system dynamics in order to better predict potential conservation threats to bivalve populations and to maximize the success of stock and disease episodes management. Cockle monitoring was conducted in 2012 and 2016, in six and eight stations, respectively, at the Ria de Aveiro coastal lagoon, Portugal. Cockles were sampled in one single occasion in 2012 and seasonally in 2016. The tested hypothesis is that the trematode community in cockles was spatially and seasonally heterogeneous but stable over time. The main result showed that despite a relative homogeneity of the parasite community structure in cockles, the among-years heterogeneity of trematode communities was higher than among-stations and among-seasons heterogeneity rejecting the postulated hypothesis. Results demonstrated that trematode communities from the Ria de Aveiro are characterized by low abundance, which resulted in a spatial and seasonal trematode homogeneity (despite an overall channel difference and a slight downstream-upstream gradient). The interannual analysis showed a worrisome loss of trematode diversity and prevalence which consequently indicates an important loss of overall diversity and/or environmental conditions reflecting the negative effects of global change (mean temperature rise and overharvesting, among others). The present study highlighted the importance of trematodes in characterising their associated environment and respective biodiversity which might be helpful to assess ecosystem ecological status and to identify threatened areas.

Seasonal variation of transcriptomic and biochemical parameters of cockles (Cerastoderma edule) related to their infection by trematode parasites.

Bivalve populations are controlled by several biotic and abiotic factors. Parasitism is among the biotic factors but is often neglected. In the present study, we focused on the transcriptomic and biochemical responses of Cerastoderma edule when parasitized as first intermediate host by the trematode Bucephalus minimus (sporocyst, the most damaging stage), and taking into account seasonal patterns. In order to test the hypothesis that the presence of B. minimus compromises cockle regular gene expression and biochemical performance and increases their vulnerability to other parasite species infection, cockles were sampled every other month during one year in Arcachon Bay (French Atlantic coast). Overall, results showed that B. minimus induced its first intermediate host defence mechanism against oxidative stress (mainly at gene level), increased host metabolism and energy demand especially in summer (revealed at both gene and biochemical level, although without significant differences) and was accompanied by a higher metacercariae abundance. Results allowed to accept the posted hypothesis and to conclude that transcriptomic and biochemical markers can provide additional and ecologically relevant information about parasite effects on their hosts, reflecting the invasion effects of pathogens but also the environmental conditions that animals experience.

Blocking of CD1d Decreases Trypanosoma cruzi-Induced Activation of CD4-CD8- T Cells and Modulates the Inflammatory Response in Patients With Chagas Heart Disease.

The control of inflammatory responses to prevent the deadly cardiac pathology in human Chagas disease is a desirable and currently unattained goal. Double-negative (DN) T cells are important sources of inflammatory and antiinflammatory cytokines in patients with Chagas heart disease and those with the indeterminate clinical form of Chagas disease, respectively. Given the importance of DN T cells in immunoregulatory processes and their potential as targets for controlling inflammation-induced pathology, we studied the involvement of CD1 molecules in the activation and functional profile of Trypanosoma cruzi-specific DN T cells. We observed that parasite stimulation significantly increased the expression of CD1a, CD1b, CD1c, and CD1d by CD14(+) cells from patients with Chagas disease. Importantly, among the analyzed molecules, only CD1d expression showed an association with the activation of DN T cells, as well as with worse ventricular function in patients with Chagas disease. Blocking of CD1d-mediated antigen presentation led to a clear reduction of DN T-cell activation and a decrease in the expression of interferon γ (IFN-γ) by DN T cells. Thus, our results showed that antigen presentation via CD1d is associated with activation of DN T cells in Chagas disease and that CD1d blocking leads to downregulation of IFN-γ by DN T cells from patients with Chagas heart disease, which may be a potential target for preventing progression of inflammation-mediated dilated cardiomyopathy.

High interleukin 17 expression is correlated with better cardiac function in human Chagas disease.

This study was designed to investigate whether the expression of interleukin 17 (IL-17) is associated with the indeterminate or cardiac clinical forms of Chagas disease and whether IL-17 expression can be correlated with patients' cardiac function. Our results demonstrated that cardiac Chagas patients have a lower intensity of expression of IL-17 by total lymphocytes and lower frequency of circulating T helper 17 cells. Correlative analysis showed that high IL-17 expression was associated with better cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. Therefore, IL-17 expression can be a protective factor to prevent myocardial damage in human Chagas disease.

Trace elements assessment in Cerastoderma glaucum from port areas in the Tunisian Mediterranean coast: The influence of parasites on bioaccumulation.

In the present study, the seasonal concentration of seven trace elements was investigated in sediment and the cockle Cerastoderma glaucum tissues from two port areas in the North-East and South of Tunisia comparing cockles non-parasitized (NP) and parasitized (P) with digenean parasites. Elements concentration in sediments analyzed in both sites revealed that Zinc (Zn), Chromium (Cr), and Lead (Pb) were the most abundant ones, while Cadmium (Cd) and Mercury (Hg) were less abundant. The bioaccumulation of trace metals and Arsenic (As) in the tissues of cockles seems to be modulated by both the infection state and the parasite species. The relationship between bioaccumulation of metals and As, trematode species and abiotic parameters showed that the availability of certain metals for uptake by P cockles of both sites was influenced by the salinity and temperature of the water. Our results corroborate the possibility of using digenean infecting bivalves in biomonitoring aquatic ecosystems.

Do Freshwater and Marine Bivalves Differ in Their Response to Wildfire Ash? Effects on the Antioxidant Defense System and Metal Body Burden.

Wildfires constitute a source of contamination to both freshwater and marine ecosystems. This study aimed to compare the antioxidant defense response of the freshwater clam Corbicula fluminea and the marine cockle (Cerastoderma edule) to wildfire ash exposure and the concomitant metal body burden. Organisms were exposed to different concentrations (0%, 12.5%, 25%, 50%, and 100%) of aqueous extracts of Eucalypt ash (AEAs) from a moderate-to-high severity wildfire. The activity of various enzymes, as well as lipid peroxidation, protein content, and metal body burden, were determined after 96 h of exposure. A significant increase in the protein content of soft tissues was observed for C. edule at AEA concentrations ≥ 25%, unlike for C. fluminea. Similarly, significant effects on lipid peroxidation were observed for cockles, but not for clams. For both species, a significant effect in the total glutathione peroxidase activity was observed at AEA concentrations ≥ 25%. Relative to the control, AEAs-exposed clams showed higher Cd content, whereas AEAs-exposed cockles showed higher Cu content, thus exhibiting different responses to the exposure to wildfire ash. The susceptibility of bivalves to ashes, at environmentally relevant concentrations, raises concern about the effects of post-fire runoff to bivalve species.

Genetic diversity of Toxoplasma gondii in South America: occurrence, immunity, and fate of infection.

Toxoplasma gondii is an intracellular parasite with a worldwide distribution. Toxoplasma gondii infections are of great concern for public health, and their impact is usually most severe in pregnant women and their foetuses, and in immunocompromised individuals. Displaying considerable genetic diversity, T. gondii strains differ widely according to geographical location, with archetypal strains predominantly found in the Northern Hemisphere and non-archetypal (atypical) strains, with highly diverse genotypes, found mainly in South America. In this review, we present an overview of the identification and distribution of non-archetypal strains of T. gondii. Special attention is paid to the strains that have been isolated in Brazil, their interaction with the host immunological response, and their impact on disease outcomes. The genetic differences among the strains are pivotal to the distinct immunological responses that they elicit. These differences arise from polymorphisms of key proteins released by the parasite, which represent important virulence factors. Infection with divergent non-archetypal strains can lead to unusual manifestations of the disease, even in immunocompetent individuals.

Bioaccessibility and oral immunization efficacy of a chimeric protein vaccine against Ascaris suum.

Human ascariasis has been characterized as the most prevalent neglected tropical disease worldwide. There is an urgent need for search to alternative prevention and control methods for ascariasis. Here we aimed to establish a protocol of oral immunization with a previously described chimera protein capable of resist through digestion and induce mucous protection against Ascaris suum infection. Mice were oral immunized with seven doses with one day interval and challenged with A. suum ten days after the last dose. In vitro digestion showed that 64% of chimeric protein was bioaccessible for absorption after digestion. Immunized mice display 66,2% reduction of larval burden in lungs compared to control group. In conclusion we demonstrated that oral immunization with chimera protein protects the host against A. suum larval migration leading to less pronounced histopathological lesions.

Immunological characterization of a VIR protein family member (VIR-14) in Plasmodium vivax-infected subjects from different epidemiological regions in Africa and South America.

Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). Deepening our knowledge of parasite biology and its molecular components is key to develop new tools for malaria control and elimination. This study aims to investigate and characterize a P. vivax protein (PvVir14) for its role in parasite biology and its interactions with the immune system. We collected sera or plasma from P.vivax-infected subjects in Brazil (n = 121) and Cambodia (n = 55), and from P. falciparum-infected subjects in Mali (n = 28), to assess antibody recognition of PvVir14. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. IgG1 and IgG3 most frequently contributed to anti-PvVir14 responses. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21- CD27-) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. When analyzed at a single-cell level, the B cell receptor gene hIGHV3-23 was only seen in subjects with active P.vivax infection where it comprised 20% of V gene usage. Among T cells, CD4+ and CD8+ levels differed (lower and higher, respectively) between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. Specific B cell subsets, anti-PvVir14 circulating antibodies, and NKT cell levels declined after treatment of P. vivax. This study provides the immunological characterization of PvVir14, a unique P. vivax protein, and possible association with acute host's immune responses, providing new information of specific host-parasite interaction. Trial registration: TrialClinicalTrials.gov Identifier: NCT00663546 & ClinicalTrials.gov NCT02334462.

Chronic infection by atypical Toxoplasma gondii strain induces disturbance in microglia population and altered behaviour in mice.

Toxoplasma gondii chronic infection is characterized by the establishment of tissue cysts in the brain and increased levels of IFN-γ, which can lead to brain circuitry interference and consequently abnormal behaviour in mice. In this sense, the study presented here sought to investigate the impact of chronic infection by two T. gondii strains in the brain of infection-resistant mice, as a model for studying the involvement of chronic neuroinflammation with the development of behavioural alterations. For that, male BALB/c mice were divided into three groups: non-infected (Ni), infected with T. gondii ME49 clonal strain (ME49), and infected with TgCkBrRN2 atypical strain (CK2). Mice were monitored for 60 days to establish the chronic infection and then submitted to behavioural assessment. The enzyme-linked immunosorbent assay was used for measurement of specific IgG in the blood and levels of inflammatory cytokines and neurotrophic factors in the brain, and the cell's immunophenotype was determined by multiparametric flow cytometry. Mice infected with ME49 clonal strain displayed hyperlocomotor activity and memory deficit, although no signs of depressive- and/or anxiety-like behaviour were detected; on the other hand, chronic infection with CK2 atypical strain induced anxiety- and depressive-like behaviour. During chronic infection by CK2 atypical strain, mice displayed a higher number of T. gondii brain tissue cysts and inflammatory infiltrate, composed mainly of CD3+ T lymphocytes and Ly6Chi inflammatory monocytes, compared to mice infected with the ME49 clonal strain. Infected mice presented a marked decrease of microglia population compared to non-infected group. Chronic infection with CK2 strain produced elevated levels of IFN-γ and TNF-ɑ in the brain, decreased NGF levels in the prefrontal cortex and striatum, and altered levels of fractalkine (CX3CL1) in the prefrontal cortex and hippocampus. The persistent inflammation and the disturbance in the cerebral homeostasis may contribute to altered behaviour in mice, as the levels of IFN-γ were shown to be correlated with the behavioural parameters assessed here. Considering the high incidence and life-long persistence of T. gondii infection, this approach can be considered a suitable model for studying the impact of chronic infections in the brain and how it impacts in behavioural responses.