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Infection with helminth parasites affects more than 1.5 billion people and is concentrated in global areas of extreme poverty, having a significant impact on public health, social life and the economy. Upon entry into the host, helminth parasites often migrate through specific tissues triggering host immunity. The immune response triggered by helminth infections is complex and depends on parasite load, site of infection, acuteness/chronicity of the infection and is species-dependent. In general, susceptibility or resistance to the infection involves the participation of the innate immune response and then the balance between several effector CD4

The leishmaniases are a group of diseases caused by the protozoan parasite belonging to the genus Leishmania. In the New World, although dogs are considered the main parasite reservoir, in the last two decades, several studies have confirmed the role of cats (Felis catus) in the epidemiology of the disease and feline leishmaniasis (FeL) is now considered to be an emerging disease. The present review summarizes the current knowledge about FeL, focusing on important immunopathological aspects, epidemiology, and diagnostic methods applied for felines in Brazil. Cats are infected with the same species of Leishmania found in dogs (i.e., Leishmania infantum). Like dogs, skin lesions are the most common in cats with clinical FeL, mainly affecting the cephalic region and less frequently the legs which may be accompanied by generalized signs or visceral involvement. Information on the immune response of cats to Leishmania infection is scarce; however, efficient infection control is seen in most cases. For diagnosis, generally, the same methods as those in dogs are used, mainly serological tools. But there is a lack of studies focusing the performance of these methods for diagnosing FeL. The estimated overall prevalence of FeL in Brazil is 8%, with L. infantum being the most prevalent species. However, infections with Leishmania braziliensis and Leishmania amazonensis have also been reported. In conclusion, although there has been an increase in the publication related to FeL in Brazil in recent years, there is a lack of research relating immune response and diagnosis of these animals. Cats have been shown to be competent hosts for Leishmania parasites, and their role in the epidemiology of the disease cannot be underestimated, especially in areas of Brazil where the disease is historically endemic.

An estimated 400 million people are infected by parasites of the genus Ascaris and the existing control measures are inefficient. Vaccine development using B cell antigens is a promising strategy for increased protection against this parasite. The present study aimed at developing a chimeric protein capable of conferring protection against infection by Ascaris sp. For this purpose, we performed B-cell epitope predictions on previously described vaccine candidate proteins from Ascaris suum and the corresponding peptides were used to construct a chimeric protein. Female BALB / c mice were immunized subcutaneously in three doses at 10 day intervals with a vaccine formulation comprised of the chimeric protein together with monophosphoryl lipid A (MPLA). Control groups included protein alone, MPLA, or PBS. After challenge infection, animals vaccinated with chimeric protein plus MPLA showed a reduction of 73.54% of larval load in the lung compared to control group animals. Animals immunized with chimeric protein plus MPLA also display higher IgG response and a reduction in lung inflammation. Our study highlights how chimeric proteins containing more than one B cell epitope can enhance immune protection against helminthic infection and offer new approaches to the development of Ascaris vaccines.

IL-17 is a cytokine produced by innate and acquired immunity cells that have an action against fungi and bacteria. However, its action in helminth infections is unclear, including in

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

Ascariasis is a neglected tropical disease that is widespread in the world and has important socioeconomic impacts. The presence of various stages of worm development in the pulmonary and intestinal mucosae induces a humoral and cellular immune response. However, although there is much evidence of the protective role of mucosal immunity against various pathogens, including helminths, there is still a gap in the knowledge about the immune response and the mechanisms of action that are involved in protection against diseases, especially in the initial phase of ascariasis. Thus, the aim of this study was to evaluate the kinetic aspects of the immune parasitological parameters in intestinal and pulmonary mucosae in male mice with early ascariasis. Therefore, two mouse strains that showed different susceptibilities to ascariasis (BALB/c and C57BL/6J) when experimentally infected with 2,500 infective eggs of Ascaris suum from time point 0 were examined: the immune parasitological parameters were evaluated each 2 days after infection over a period of 12 days. The results were suggestive of a synergetic action of intestinal and pulmonary secretory IgA (S-IgA) contributing to protection against early ascariasis by reducing the amount of migrating larvae as well as the influx of leukocytes in the lung and the consequent impairment of pulmonary capacity.

BACKGROUND Interactions between parasites during coinfections are often complex and can impact immunization and treatment programs, as well as disease outcomes and morbidity. However, little is known about these interactions and the mechanisms involved. METHODS In this study, a coproparasitological survey was carried out in school-age children living in endemic areas of parasitic infection in the state of Sergipe, in Northeastern Brazil. Anti-helminth-specific and total secretory immunoglobulin-A (SIgA) levels were measured in stool and saliva samples and were compared in children presenting monoparasitism, polyparasitism (helminths and/or intestinal protozoa), and no infections. RESULTS The survey showed that protozoa were more prevalent than helminths, and that there was a high frequency of polyparasitism in the studied population, mainly from combinations of protozoan species. Although less frequent, combinations between species of protozoa and helminths were also observed. The levels of salivary SIgA in these co-infected individuals were lower than the average observed in infections with helminths alone. CONCLUSIONS Although the children participating in this survey were asymptomatic, and it was, therefore, not possible to evaluate the impact of salivary SIgA reduction on the diseases, the study highlights the need for further investigations of coinfections by intestinal parasites and the effects on immune response induced by the interactions between different parasites.

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host’s organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles., Author summary Ascariasis is the most prevalent but neglected tropical disease in the world, affecting more than 450 million people, mainly children. The parasites life cycle can be divided into the following two distinct phases after the initial infection: (i) the first stage called larval ascariasis involves migration of parasitic larval stages through several tissues (intestinal mucosa, blood circulation, liver, and lung/airways) and is marked by mechanical injuries, followed by an intense local inflammatory response, (ii) the second involves the establishment of adult worms in the lumen of the small intestine, causing the despoilment of nutrients and secretory/excretory parasitic products that downmodulate host immune response and characterize the chronic infection. Innate and adaptive immune responses are intrinsically connected and their cross-talk is very important for host homeostasis and is crucial in dealing with helminthiasis. In this study, we evaluated the importance of eosinophils mediating IgA production in mucosal sites, tissue inflammation, and remodeling, as well as controlling parasite burden during experimental larval ascariasis. This study provides a new perspective suggesting that stimulation by probiotics may prevent the lung pathology associated with ascariasis.

Made available in DSpace on 2022-04-29T08:38:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Rationale: The FDA-approved Dimethyl Fumarate (DMF) as an oral drug for Multiple Sclerosis (MS) treatment based on its immunomodulatory activities. However, it also caused severe adverse effects mainly related to the gastrointestinal system. Objective: Investigated the potential effects of solid lipid nanoparticles (SLNs) containing DMF, administered by inhalation on the clinical signs, central nervous system (CNS) inflammatory response, and lung function changes in mice with experimental autoimmune encephalomyelitis (EAE). Materials and methods: EAE was induced using MOG35-55 peptide in female C57BL/6J mice and the mice were treated via inhalation with DMF-encapsulated SLN (CTRL/SLN/DMF and EAE/SLN/DMF), empty SLN (CTRL/SLN and EAE/SLN), or saline solution (CTRL/saline and EAE/saline), every 72 h during 21 days. Results: After 21 days post-induction, EAE mice treated with DMF-loaded SLN, when compared with EAE/saline and EAE/SLN, showed decreased clinical score and weight loss, reduction in brain and spinal cord injury and inflammation, also related to the increased influx of Foxp3+ cells into the spinal cord and lung tissues. Moreover, our data revealed that EAE mice showed signs of respiratory disease, marked by increased vascular permeability, leukocyte influx, production of TNF-α and IL-17, perivascular and peribronchial inflammation, with pulmonary mechanical dysfunction associated with loss of respiratory volumes and elasticity, which DMF-encapsulated reverted in SLN nebulization. Conclusion: Our study suggests that inhalation of DMF-encapsulated SLN is an effective therapeutic protocol that reduces not only the CNS inflammatory process and disability progression, characteristic of EAE disease, but also protects mice from lung inflammation and pulmonary dysfunction. Neuroscience Group Department of Physiology and Biophysics Institute of Biological Sciences Federal University of Minas Gerais (UFMG), MG Nanoneurobiophysics Research Group Department of Physics Chemistry and Mathematics Federal University of Sao Carlos (UFSCAR), Sao Paulo State of Sao Paulo University (UNESP) Drugs and Medicines Department School of Pharmaceutical Sciences, Sao Paulo Laboratory of Pulmonary Immunology and Mechanics Department of Physiology and Biophysics Institute of Biological Sciences Federal University of Minas Gerais (UFMG), MG Laboratory of Immunology and Genomics of Parasites Department of Parasitology Institute of Biological Sciences Federal University of Minas Gerais (UFMG), MG Center for Technology and Research in Magneto-Resonance (CTPMAG) Federal University of Minas Gerais (UFMG), MG Department of Pathology Institute of Biological Sciences Federal University of Minas Gerais (UFMG), MG Laboratory of Functional Genetics Department of Genetics Ecology and Evolution Institute of Biological Sciences Federal University of Minas Gerais (UFMG), MG Department of Pharmacology Institute of Biological Sciences Federal University of Goias (UFG), GO State of Sao Paulo University (UNESP) Drugs and Medicines Department School of Pharmaceutical Sciences, Sao Paulo

Background Ascariasis and malaria are highly prevalent parasitic diseases in tropical regions and often have overlapping endemic areas, contributing to high morbidity and mortality rates in areas with poor sanitary conditions. Several studies have previously aimed to correlate the effects of Ascaris-Plasmodium coinfections but have obtained contradictory and inconclusive results. Therefore, the present study aimed to investigate parasitological and immunopathological aspects of the lung during murine experimental concomitant coinfection by Plasmodium berghei and Ascaris suum during larvae ascariasis. Methods C57BL/6J mice were inoculated with 1 × 104P. berghei strain NK65-NY-infected red blood cells (iRBCs) intraperitoneally and/or 2500 embryonated eggs of A. suum by oral gavage. P. berghei parasitaemia, morbidity and the survival rate were assessed. On the seventh day postinfection (dpi), A. suum lung burden analysis; bronchoalveolar lavage (BAL); histopathology; NAG, MPO and EPO activity measurements; haematological analysis; and respiratory mechanics analysis were performed. The concentrations of interleukin (IL)-1β, IL-12/IL-23p40, IL-6, IL-4, IL-33, IL-13, IL-5, IL-10, IL-17A, IFN-γ, TNF and TGF-β were assayed by sandwich ELISA. Results Animals coinfected with P. berghei and A. suum show decreased production of type 1, 2, and 17 and regulatory cytokines; low leukocyte recruitment in the tissue; increased cellularity in the circulation; and low levels of NAG, MPO and EPO activity that lead to an increase in larvae migration, as shown by the decrease in larvae recovered in the lung parenchyma and increase in larvae recovered in the airway. This situation leads to severe airway haemorrhage and, consequently, an impairment respiratory function that leads to high morbidity and early mortality. Conclusions This study demonstrates that the Ascaris-Plasmodium interaction is harmful to the host and suggests that this coinfection may potentiate Ascaris-associated pathology by dampening the Ascaris-specific immune response, resulting in the early death of affected animals.

Toxocariasis is a neglected disease that affects people around the world. Humans become infected by accidental ingestion of eggs containing Toxocara canis infective larvae, which upon reaching the intestine, hatch, penetrate the mucosa and migrate to various tissues such as liver, lungs and brain. Studies have indicated that Th2 response is the main immune defense mechanism against toxocariasis, however, there are still few studies related to this response, mainly the IL-33/ST2 pathway. Some studies have reported an increase in IL-33 during helminth infections, including T. canis. By binding to its ST2 receptor, IL-33 stimulating the Th2 polarized immune cell and cytokine responses. Thus, we aimed to investigate the role of the IL-33/ST2 pathway in the context of T. canis larval migration and the immunological and pathophysiological aspects of the infection in the liver, lungs and brain from Wild-Type (WT) BALB/c background and genetically deficient mice for the ST2 receptor (ST2-/-). The most important findings revealed that the IL-33/ST2 pathway is involved in eosinophilia, hepatic and cerebral parasitic burden, and induces the formation of granulomas related to tissue damage and pulmonary dysfunction. However, ST2-/- mice, the immune response was skewed to Th1/Th17 type than Th2, that enhanced the control of parasite burden related to IgG2a levels, tissue macrophages infiltration and reduced lung dysfunction. Collectively, our results demonstrate that the Th2 immune response triggered by IL-33/ST2 pathway mediates susceptibility to T. canis, related to parasitic burden, eosinophilia and granuloma formation in which consequently contributes to tissue inflammation and injury., Author summary Toxocariasis is a neglected disease caused by Toxocara canis, which has 19% worldwide seroprevalence, and is associated with socioeconomic, geographic and environmental factors. Humans become infected by accidental ingestion of T. canis eggs present in contaminated food, water or soil. After ingestion, the larvae hatch in the intestine and can reach various tissues such as liver, lung and brain. Helminth infections usually trigger a Th2 immune response in the host, by releasing cytokines such as IL-4, IL-5, IL-13 and IL-33. IL-33 is an alarmin that binds to the ST2 receptor, and some studies have observed an increase in this cytokine in toxocariasis, however there are no studies regarding the IL-33/ST2 role in this infection. Thus, we evaluated the influence of this pathway by analyzing immunological and pathophysiological aspects in T. canis-infected mice. Our results demonstrated that the IL-33/ST2 pathway is related to parasite burden on the liver and brain and also increases the number of eosinophils in the blood and tissues. In addition, it involved with the pulmonary immune response and granulomas with impact in lung function. In conclusion, the IL-33/ST2 pathway governs the host susceptibility to T. canis in mice.

Apoptosis is associated with resolution of inflammation. However, apoptosis may also occur in active inflammation, balancing inflammatory recruitment instead of a resolution event. To test that hypothesis, we measured apoptosis and chemokines expression, involved in recruitment of inflammatory cells. Clinical affected and subclinically infected dogs with canine leishmaniosis (CanL) and uninfected controls were assessed. Apoptosis in renal tissue (glomeruli, tubules, and inflammatory infiltrate) and cellularity in inflammatory foci were quantified. Messenger RNA of CCL5, CCL4, MCP-1, MCP-2, Caspase (Casp) 3, Casp 8, Casp 9, Bax, Bcl2 and Fas were quantified by qRT PCR. Clinical affected dogs showed more intense inflammation and higher cellularity in the inflammatory infiltrates than subclinically infected ones, which were higher than controls. Glomerular and tubular cells showed higher apoptotic index in clinical affected dogs when compared to controls. Apoptosis within the inflammatory infiltrates was higher in clinical affected dogs. Bax/Bcl2 ratio and CCL4 showed higher expression in kidney from clinical affected when compared to subclinically infected dogs. Casp 3/CCL4 ratio expression were higher in subclinically infected dogs than in the clinical affected group. Additionally, results suggest that Casp 3/CCL4 ratio is balancing towards an inflammatory recruitment and CCL4 and Bax/Bcl2 ratio expression is associated with active inflammation in clinical affected CanL. Data demonstrate that apoptosis was not always correlated with resolution of inflammation, when a morphometric and a molecular evaluation were performed concomitantly. In kidneys of Leishmania infected dogs, apoptosis and chemokines may be balancing inflammatory recruitment. In conclusion, Bax/Bcl2 ratio, chemokines, Casp 8, Casp 3 and Fas were associated with renal apoptosis, active inflammation and increased inflammatory recruitment observed in clinical affected animals, influencing the clinical presentation of leishmaniosis.

The poor immunogenicity displayed by some antigens has encouraged the development of strategies to improve the immune response and safety of vaccine candidates, resulting in an intense search for substances that potentiate vaccine response. Adjuvants have these properties helping vaccine candidates to induce a strong, durable, and fast immune response. In this study, we evaluated the specific immune response of adjuvants alone, Saponin (SAP), Incomplete Freund’s Adjuvant (IFA) and Monophosphoryl lipid-A SE (MPL-SE®) and in combination with total antigen of L. braziliensis (LB): LBSAP, LBIFA and LBMPL. The specific immune response induced by these compositions demonstrated that they were powerfully immunogenic, increasing cellular infiltration in the skin. Draining lymph nodes cultures showed that LBIFA and LBMPL have higher ability to increase the capacity of APCs to present antigens, with increased frequency of CD11c+CD86+ cells. SAP, MPL, LBSAP, LBIFA and LBMPL could activate lymphocytes increasing expression of CD69 and CD25. LBSAP group was an excellent inducer of pro-inflammatory cytokines at 24 h. At 48 h, higher cytokines production was observed in IFA, LBIFA, MPL and LBMPL groups. Our data demonstrate that LBSAP and LBMPL are potential formulations to be tested in other experimental models. Also, the data obtained could expand the knowledge about immune response after sensitization and also contribute to the development of safe, immunogenic and effective vaccines.

This study investigates the relationship between helminth infection and allergic sensitization by assessing the influence of preexisting allergy on the outcome of helminth infections, rather than the more traditional approach in which the helminth infection precedes the onset of allergy. Here we used a murine model of house dust mite–induced (HDM-induced) allergic inflammation followed by Ascaris infection to demonstrate that allergic sensitization drives an eosinophil-rich pulmonary type 2 immune response (Th2 cells, M2 macrophages, type 2 innate lymphoid cells, IL-33, IL-4, IL-13, and mucus) that directly hinders larval development and reduces markedly the parasite burden in the lungs. This effect is dependent on the presence of eosinophils, as eosinophil-deficient mice were unable to limit parasite development or numbers. In vivo administration of neutralizing antibodies against CD4 prior to HDM sensitization significantly reduced eosinophils in the lungs, resulting in the reversal of the HDM-induced Ascaris larval killing. Our data suggest that HDM allergic sensitization drives a response that mimics a primary Ascaris infection, such that CD4(+) Th2-mediated eosinophil-dependent helminth larval killing in the lung tissue occurs. This study provides insight into the mechanisms underlying tissue-specific responses that drive a protective response against the early stages of the helminths prior to their establishing long-lasting infections in the host.

Human ascariasis is one of the most prevalent neglected tropical diseases worldwide. The immune response during human ascariasis is characterized by Th2 polarization and a mixed Th2/Th17 response during the pathogenesis of experimental larval ascariasis. Cytokines and other pro-inflammatory mediators, such as nitric oxide (NO), are involved in helminthic infections. However, the role of NO in ascariasis remains unclear.Given the importance of NO in inflammation, we aimed to determine the immunological and histopathological alterations in the livers of C57BL/6 iNOSIn this study, parasitic load was evaluated in the livers of wild type C57BL/6 and C57BL/6 iNOSThe results showed that NO is important for controlling parasitic load during infection by A. suum. C57BL/6iNOSWe demonstrated that NO is a crucial inflammatory molecule during Ascaris sp. infection and controls the establishment of the parasite and the development of the host immune response in the liver.

Visceral leishmaniasis (VL) is an important public health problem in the world, and control measures are insufficient to avoid the spread of this neglected disease. Dogs are important domestic reservoirs of Leishmania parasites in countries where VL is a zoonosis, representing a major source of infection between sand fly vectors and humans. In this context, a precise diagnosis of canine leishmaniasis (CanL) could help to reduce the number of human cases. Distinct approaches for the diagnosis of CanL have used recombinant proteins in serological assays. However, variable results of the antigens have been found, mainly to diagnosis asymptomatic cases. The present study used bioinformatics to select specific B-cell epitopes of four Leishmania infantum proteins, which had previously been proven to be antigenic in VL, aiming to produce a novel chimeric protein and to evaluate it for the diagnosis of CanL. Seven B-cell epitopes were identified and used to construct the chimera, which was analyzed in a recombinant format through an ELISA assay against a canine serological panel. A soluble Leishmania antigenic extract (SLA) was used as an antigen control. Results showed 100 % sensitivity and specificity for chimera, while when using SLA the values were 26.0 % and 96.4 %, respectively. The performance of chimera was compared with a commercial kit using asymptomatic and symptomatic dog sera, and the data showed that no false-negative result was found when the recombinant protein was used. However, when using the commercial kit, 40.0 % and 16.0 % of the false-negative results were found, respectively. In conclusion, the recombinant chimera showed an antigenic potential to be evaluated in new studies against a larger serological panel for the diagnosis of CanL.

Human trypanosomiases and animal trypanosomoses are caused by distinct protozoan parasites of the genus Trypanosoma. The etiological agents of bovine trypanosomosis (BT) are T. vivax, T. congolense, or T. brucei, whose acute infections are initially characterized by hyperthermia, following moderate to severe anemia, subcutaneous edema, lethargy, reduced milk production, progressive weight loss, enlarged lymph nodes, reproductive disorders and death. Animals that survive the acute phase might recover and progress to the chronic, often asymptomatic, phase of infection. Despite their low sensitivity due to the characteristic low parasitemia, simple and costless direct parasitological examinations are the preferred diagnostic methods for animals. Thus, most of the epidemiological studies of BT are based on serological techniques using crude antigen. In this study, we describe the use of the MyxoTLm recombinant protein as an antigen on serological assays. Anti-T. vivax IgM and anti-T. vivax IgG ELISA assays using purified MyxoTLm revealed specificity rates of 91.30 % and 95.65 % and sensitivity rates of 82.35 % and 88.23 %, respectively, being higher than reported for crude antigens. Also, MyxoTLm demonstrated a good performance to detect IgM (ROC curve area = 0.8568) and excellent performance to detect IgG (ROC curve area = 0.9565) when compared to a crude antigen. T. evansi crude antigen used in the indirect anti-T. vivax IgM ELISA reached 70.58 % sensitivity and 78.26 % specificity, and had a lower test performance (ROC curve area = 0.7363). When applied to the anti-T. vivax IgG ELISA, the crude antigen reached 82.35 % sensitivity and 69.56 % specificity, also presenting a low performance with area under the ROC curve of 0.7570. Therefore, the use of MyxoTLm as an antigen on serological diagnosis of BT revealed to increase the sensitivity and the specificity if compared to crude antigens.

Treatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection.

Ascaris lumbricoides and Ascaris suum are two closely related parasites that infect humans and pigs. The zoonotic potential of A. suum has been a matter of debate for decades. Here we sought to investigate the potential human infection by A. suum and its immunological alterations. We orally infected five healthy human subjects with eggs embraced by A. suum. The infection was monitored for symptoms and possible respiratory changes, by an interdisciplinary health team. Parasitological, hematological analyses, serum immunoglobulin, cytokine profiles, and gene expression were evaluated during the infection. Our results show that A. suum is able to infect and complete the cycle in humans causing A. lumbricoides similar symptoms, including, cough, headache, diarrhea, respiratory discomfort and chest x-ray alterations coinciding with larvae migration in the lungs. We also observed activation of the immune system with production of IgM and IgG and a Th2/Th17 response with downregulation of genes related to Th1 and apoptosis. PCA (Principal componts analysis) show that infection with A. suum leads to a change in the immune landscape of the human host. Our data reinforce the zoonotic capacity of A. suum and bring a new perspective on the understanding of the immune response against this parasite.

The complexity and multifactorial characteristics of Chagas disease pathogenesis hampers the establishment of appropriate experimental/epidemiological sets, and therefore, still represents one of the most challenging fields for novel insights and discovery. In this context, we used a set of attributes including phenotypic, functional and serological markers of immune response as candidates to decode the genotype-specific immune response of experimental T. cruzi infection. In this investigation, we have characterized in C57BL/6 J mice, the early (parasitemia peak) and late (post-parasitemia peak) aspects of the immune response elicited by T. cruzi strains representative of TcI, TcII or TcVI. The results demonstrated earlier parasitemia peak for TcII/Y strain followed by TcVI/CL-Brener and TcI/Colombiana strains. A panoramic overview of phenotypic and functional features of the TCD4+, TCD8+ and B-cells from splenocytes demonstrated that mice infected with TcI/Colombiana strain exhibited at early stages of infection low levels of most cytokine+ cells with a slight increase at late stages of infection. Conversely, mice infected with TcII/Y strain presented an early massive increase of cytokine+ cells, which decreases at late stages. The TcVI/CL-Brener strain showed an intermediate profile at early stages of infection with a slight increase later on at post-peak of parasitemia. The panoramic analysis of immunological connectivity demonstrated that early after infection, the TcI/Colombiana strain trigger immunological network characterized by a small number of connectivity, selectively amongst cytokines that further shade towards the late stages of infection. In contrast, the TcII/Y strain elicited in more imbricate networks early after infection, comprising a robust number of interactions between pro-inflammatory mediators, regulatory cytokines and activation markers that also decrease at late infection. On the other hand, the infection with TcVI/CL-Brener strain demonstrated an intermediate profile with connectivity axes more stable at early and late stages of infection. The analysis of IgG2a reactivity to AMA, TRYPO and EPI antigens revealed that at early stages of infection, the genotype-specific reactivity to AMA, TRYPO and EPI to distinguish was higher for TcI/Colombiana as compared to TcII/Y and TcVI/CL while, at late stages of infection, higher reactivity to AMA was observed in mice infected with TcVI/CL and TcII/Y strains. The novel systems biology approaches and the use of a flow cytometry platform demonstrated that distinct T. cruzi genotypes influenced in the phenotypic and functional features of the host immune response and the genotype-specific serological reactivity during early and late stages of experimental T. cruzi infection.

This study aimed to evaluate the performance of the point-of-care circulating cathodic antigen (POC-CCA) test in a highly endemic area in Brazil, comparing it to the Kato-Katz (KK) technique for sensitivity, specificity and the intensity of the reaction of the test in relation to the parasitic load. The community in Sergipe, Brazil, participated in the study, providing three stool samples, one of urine (POC-CCA) and fingers tick blood sample was tested by enzyme-linked immunosorbent assay (ELISA). Sensitivity, specificity, positive predictive value, negative predictive value, accuracy, kappa coefficient and Spearman's correlation were calculated for the POC-CCA test using the KK as the reference. The prevalence of schistosomiasis by KK testing was 48.82%; POC-CCA (t+) 66.14%; POC-CCA (t−) 45.24%. ELISA results showed 100% agreement in individuals with high and moderate eggs per gram (EPG). POC-CCA presented good diagnostic performance in individuals with medium and high EPG, but there were a high number of false negatives in individuals with low intensity infections. As observed, POC-CCA-filter test improves accuracy and sensitivity compared to a conventional test.

Visceral leishmaniasis (VL) is a systemic parasitic disease that leads to high rates of morbidity and mortality in humans worldwide. There is a great need to develop new drugs and novel strategies to make chemotherapy for this disease more efficacious and well tolerated. Recent reports on the immunomodulatory effects and the low toxicity of the spherical carbon nanostructure fullerol led us to investigate in vitro and in vivo antileishmanial activity in free and encapsulated forms in liposomes. When assayed against intramacrophagic Leishmania amastigotes, fullerol showed a dose-dependent reduction of the infection index with IC50 of 0.042 mg/mL. When given daily by i.p. route for 20 days (0.05 mg/kg/d) in a murine model of acute VL, fullerol promoted significant reduction in the liver parasite load. To improve the delivery of fullerol to the infection sites, liposomal formulations were prepared by the dehydration-rehydration method. When evaluated in the acute VL model, liposomal fullerol (Lip-Ful) formulations given i.p. at 0.05 and 0.2 mg/kg with 4-days intervals were more effective than the free form, with significant parasite reductions in both liver and spleen. Lip-Ful at 0.2 mg/kg promoted complete parasite elimination in the liver. The antileishmanial activity of Lip-Ful was further confirmed in a chronic model of VL. Lip-Ful was also found to induce secretion of pro-inflammatory TNF-α, IFN-γ and IL-1β cytokines. In conclusion, this work reports for the first time the antileishmanial activity of fullerol and introduces an innovative approach for treatment of VL based on the association of this nanostructure with liposomes.

Background Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits in host health, increases production costs, and incurs a reduced food supply. The control of these parasites is usually done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules with anthelmintic potential is necessary. Methods In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans; Ancylostoma ceylanicum; and Ascaris suum. Results The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified the major substance present in two sub-fractions of this extract as ketamine. Then, we tested this compound on C. elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a difference between the animal groups when considering the number of worms recovered from the intestines of animals treated with ketamine (6 mg) and albendazole (P > 0.05). The parasite burden of larvae recovered from the lungs of mice treated with ketamine was similar to those treated with ivermectin. Conclusions The results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo, thus confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist of a new method of controlling parasites.

The pathogenesis of Canine leishmaniosis (CanL) is associated with altered cytokine expression and parasitic tissue shows a lot of inflammation. The aim of this study was to assess the renal inflammation and cytokine expression in eight symptomatic and eight asymptomatic Leishmania- infected dogs, and seven uninfected control dogs. Kidney fragments were stained with hematoxylin and eosin for morphometric evaluation. mRNA expression levels of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, IL-4, IL-10, and IL-12 were assessed in the kidney fragments using quantitative real time-polymerase chain reaction. Inflammation, quantified by the average area of the infiltrated immune cells, was greater in symptomatic dogs than in those asymptomatic, whereas asymptomatic dogs exhibited higher inflammation than the control dogs (p0.05, Tukey's test). Expression levels of IFN-γ, TNF-α, IL-4, IL-10, and IL-12 were upregulated in symptomatic dogs and downregulated in asymptomatic dogs compared with those of the uninfected group. Furthermore, IL-4 showed higher expression in symptomatic dogs than in asymptomatic ones (p0.05, Mann-Whitney test), which was directly associated with clinical manifestations (p0.05, Chi-square test). However, IL-12 was predominantly expressed in symptomatic dogs, shifting the balance from IL-12/IL-4 to IL-12, which elicits a change in the inflammatory response. Leishmania was not found in the renal tissues in any one of the studied groups. Our data suggests that the balance between IL-12 and IL-4 plays an important role in the regulation of inflammation in renal tissue and clinical presentations in CanL.

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.

Trematodes have complex life cycles with multiple hosts. Biomphalaria snails commonly act as the first intermediate hosts of several species that can affect human and animal health. The specific identification of larval trematodes found in snails is difficult and limited, since the taxonomy of these flukes is based on morphological traits of the adults found in vertebrates. Despite recent advances worldwide, studies aiming at the use of molecular tools for the identification of cercariae found in snails are scarce in the South America. In fact, most studies are focused on Schistosoma mansoni, with few efforts directed towards the identification of larvae of other parasites found in planorbids. When reported, these other parasites are identified as cercarial types, an artificial morphological system of classification. Therefore, alternative strategies for a correct, rapid and inexpensive identification of larval trematodes found in Biomphalaria are needed. This work aimed at developing a methodology capable of distinguishing four important families of trematodes (Clinostomidae, Echinostomatidae, Schistosomatidae and Strigeidae) commonly found infecting species of Biomphalaria. Using the rDNA sequences of 34 species as input for the online tool TipMT, we designed trematode family-specific primers targeting the ITS region optimized to be used in multiplex PCR. The panel of primers identified in this study was effective at the same PCR condition. The specificity of the primers was confirmed, and the PCR sensitivity ranged from 0.1 ng to 1 ag of the DNA of the parasite. This methodology was also effective for the detection of coinfection. Through a simple, fast, accurate, and inexpensive methodology, it is possible to properly identify the trematode families included in this study in a single PCR reaction. A family level identification provides important information about probable hosts, pattern of life cycle and possible impacts that the infection generates in a specific region, thus allowing the design of better control strategies, especially for those infections that have medical and veterinary importance.

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information along with RNA Seq data to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, ‘hypothetical’ proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. SCP/TAPS proteins are immunogenic in nematode infections, so we expressed four of those identified in this study in recombinant form and showed that they are all recognized to varying degrees by serum antibodies from hookworm-infected subjects from a disease-endemic area of Brazil. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and diagnostic targets., Author summary Hookworms infect hundreds of millions of people in tropical regions of the world. Adult worms reside in the small bowel where they feed on blood, causing iron-deficiency anemia when present in large numbers and contributing substantially to the poverty in these regions. Hookworms inject excretory/secretory (ES) products into the gut tissue when they feed, and while the protein constituents of ES products have been characterized for a number of animal hookworm species, difficulty in obtaining sufficient live human hookworms has thus far precluded characterization of the secreted proteome. Herein we describe the ES proteins of the major human hookworm, Necator americanus, and utilize this information to significantly improve the available genome sequence. Almost 200 ES proteins were identified and compared to the secreted proteomes of other parasitic roundworms to provide a molecular snapshot of the host-parasite interface. We produced recombinant forms of some of the identified proteins and showed that they are all recognized to varying degrees by antibodies from hookworm-infected subjects. Our work sheds light on important families of proteins that might be key for parasite survival in the human host, and presents a dataset that can now be mined in the search for vaccine, drug and diagnostic targets.

Leishmaniasis is a neglected disease, caused by a parasite of Leishmania genus and widespread in the tropical and subtropical areas of the world. Currents drugs are limited due to their toxicity and parasite resistance. Therefore, the discovery of new treatment, more effective and less toxic, is urgent. In this study, we report the synthesis of six gem-dihydroperoxides (2a-2f), with yields ranging from 10 % to 90 %, utilizing a new methodology. The dihydroperoxides were converted into ten tetroxanes (3a-3j), among which six (3b, 3c, 3d, 3g, 3h and 3j) showed activity against intracellular amastigotes of Leishmania amazonensis. The cytotoxicity of all compounds was also evaluated against canine macrophages (DH82), human hepatoma (HepG2) and monkey renal cells (BGM). Most compounds were more active and less toxic than potassium antimonyl tartrate trihydrate, used as positive control. Amongst all tetroxanes, 3b (IC50 =0.64 μm) was the most active, being more selective than positive control in relation to DH82, HepG2 and BGM cells. In summary, the results revealed a hit compound for the development of new drugs to treat leishmaniasis.

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (, Graphical abstract Image 1, Highlights • Bacillus thuringiensis (Bt) Cry5B is a new pan-hookworm cure. • Bt Cry5B is highly active against Ancylostoma caninum hookworm infections in dogs. • Bt Cry5B is highly active against Necator americanus. • Cry5B acts independent of the host immune system and directly on hookworms. • Cry5B retains full bioactivity after repeated dosing.

The development of T follicular helper cells (Tfh) is a multifactorial process that occurs in multiple stages. After their activation the Tfh cells interact with the B cells to complete their differentiation. During this process, the Tfh cells begin to express canonical molecules such as the transcription factor B-cell lymphoma 6 protein, the CXC chemokine receptors type 5, and the inducible T-cell costimulator, as well as secreting other molecules such as IL-21. This whole process is regulated positively and negatively by several factors so that the best response is offered in the face of diseases of various origins, among them helminthiasis. In this context, the role of circulating Tfh, IL-4 and IgG subtypes is essential for an effective response against these pathogens. In this review, the migration process and the differentiation of Tfh, the regulation, their cell subtypes and the role of Tfh in the context of helminth infections will be addressed.

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis., Author summary Visceral Leishmaniasis (VL) is a neglected tropical disease caused by protozoa Leishmania infantum and L. donovani. This illness affects millions of people in various regions of the world and can be fatal if not early treated. In the Americas and Southern Europe, the VL is a zoonotic disease, being the dog its main reservoir. The early and correct diagnosis of dogs and humans infected with Leishmania is essential for the efficient disease control. However, current diagnostic methods have limitations, as they are not sufficiently sensitive and/or specific, and may not be able to identify all cases of VL or exhibit cross-reactions with other infections. We produced a recombinant protein, rKDDR-plus, constituted only by highly repetitive sequences kinesin-derived from L. infantum, and compare its performance with other kinesin-derived antigens (rK39 and rKDDR), but that have less repetitive sequences. The rKDDR-plus protein exhibited the best performance among the studied antigens, presenting a greater specificity in the VL diagnosis, both in the ELISA and rapid test platforms. Therefore, this antigen can contribute to the control of leishmaniasis in humans and dogs, through a diagnostic test with high specificity, achieved due to the use of the new rKDDR-plus antigen.

Chagas disease, caused by the parasite Trypanosoma cruzi, is considered endemic in more than 20 countries but lacks both an approved vaccine and limited treatment for its chronic stage. Chronic infection is most harmful to human health because of long-term parasitic infection of the heart. Here we show that immunization with a virus-like particle vaccine displaying a high density of the immunogenic α-Gal trisaccharide (Qβ-αGal) induced several beneficial effects concerning acute and chronic T. cruzi infection in α1,3-galactosyltransferase knockout mice. Approximately 60% of these animals were protected from initial infection with high parasite loads. Vaccinated animals also produced high anti-αGal IgG antibody titers, improved IFN-γ and IL-12 cytokine production, and controlled parasitemia in the acute phase at 8 days post-infection (dpi) for the Y strain and 22 dpi for the Colombian strain. In the chronic stage of infection (36 and 190 dpi, respectively), all of the vaccinated group survived, showing significantly decreased heart inflammation and clearance of amastigote nests from the heart tissue., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a significant endemic infectious disease in Latin America and is spreading in the U.S. and Europe with the presence of its insect transmission vector. No approved vaccine against Chagas disease exists. We describe a vaccine candidate based on a carbohydrate found on the T. cruzi cell surface, linked in the vaccine to a virus-like particle that provides a strong and focused immune response. Mice were immunized and challenged with the Trypanosoma cruzi parasites from two strains (Y and Colombian). Vaccination conferred substantial protection of mice against infection, compared with the unvaccinated group. Vaccinated animals presented low parasitemia, increased production of pro-inflammatory cytokines IL-12 and IFN-γ, decreased cardiomyocyte damage, and rapid clearance of parasite nests from heart tissue. These effects were especially significant at time points modeling chronic disease, an important consideration for this pathogen. We, therefore, believe this is a valuable path to pursue in the development of vaccines against Chagas disease.

The aim of this work was to elucidate the immunopathological mechanisms of how helminths may influence the course of a viral infection, using a murine model. Severe virulence, a relevant increase in the virus titres in the lung and a higher mortality rate were observed in Ascaris and Vaccinia virus (VACV) co-infected mice, compared with VACV mono-infected mice. Immunopathological analysis suggested that the ablation of CD8+ T cells, the marked reduction of circulating CD4+ T cells producing IFN-γ, and the robust pulmonary inflammation were associated with the increase of morbidity/mortality in co-infection and subsequently with the negative impact of concomitant pulmonary ascariasis and respiratory VACV infection for the host. On the other hand, when evaluating the impact of the co-infection on the parasitic burden, co-infected mice presented a marked decrease in the total number of migrating Ascaris lung-stage larvae in comparison with Ascaris mono-infection. Taken together, our major findings suggest that Ascaris and VACV co-infection may potentiate the virus-associated pathology by the downmodulation of the VACV-specific immune response. Moreover, this study provides new evidence of how helminth parasites may influence the course of a coincident viral infection.

Ascariasis is considered the most neglected tropical disease, and is a major problem for the public health system. However, idiopathic pulmonary fibrosis (IPF) is a result of chronic extracellular deposition of matrix in the pulmonary parenchyma, and thickening of the alveolar septa, which reduces alveolar gas exchange. Considering the high rates of ascariasis and pulmonary fibrosis, we believe that these two diseases may co-exist and possibly lead to comorbidities. We therefore investigated the mechanisms involved in comorbidity of Ascaris suum (A. suum) infection, which could interfere with the progression of pulmonary fibrosis. In addition, we evaluated whether a previous lung fibrosis could interfere with the pulmonary cycle of A. suum in mice. The most important findings related to comorbidity in which A. suum infection exacerbated pulmonary and liver injury, inflammation and dysfunction, but did not promote excessive fibrosis in mice during the investigated comorbidity period. Interestingly, we found that pulmonary fibrosis did not alter the parasite cycle that transmigrated preferentially through preserved but not fibrotic areas of the lungs. Collectively, our results demonstrate that A. suum infection leads to comorbidity, and contributes to the aggravation of pulmonary dysfunction during pulmonary fibrosis, which also leads to significant liver injury and inflammation, without changing the A. suum cycle in the lungs., Author summary Ascariasis is considered a major problem for the public health system, which has an estimated 800 million infected people worldwide. It occurs in the United States, Africa, Asia, and Latin America, and is generally associated with poverty and precarious health conditions. Pulmonary fibrosis affects 14–63 people per 100,000 habitants/year, and is characterized by collagen deposition and alveolar wall thickening. The comorbidities caused by infections are commonly associated with pulmonary fibrosis exacerbations, poor prognosis, and high mortality. Despite the comorbidities caused by helminth infections, which display a pulmonary parasitic cycle such as that of Ascaris, there is no evidence relating to pulmonary fibrosis progression, possibly because Ascariasis is considered a neglected disease. We evaluated the role of Ascaris during pulmonary fibrosis. We considered two simple questions: (1) Whether Ascaris infection could protect or aggravate fibrosis (comorbidities) and (2) whether pulmonary fibrosis could change the cycle of Ascaris as a result of increased alveolar thickening, larvae retention, and the limitation of influx into airways. We answered both questions as follows: (1) Ascaris infection exacerbates pulmonary and liver injury and inflammation, but not fibrosis; and (2) Pulmonary fibrosis did not alter the course of Ascaris cycle in lungs during transmigration into airways, because Ascaris preferentially seeks and penetrates into the lung areas, which are thought to be preserved, but not into fibrotic areas.

The main control strategy for Ascaris lumbricoides is mass drug administration (especially with benzimidazoles), which can select strains of parasites resistant to treatment. Mutations in the beta-tubulin isotype-1 gene at codons 167, 198 and 200 have been linked to benzimid azole resistance in several nematodes. The mutation in codon 200 is the most frequent in different species of parasites, as previously observed in Necator americanus and Trichuris trichiura; however, this mutation has never been found in populations of A. lumbricoides. This study aimed to screen for single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codon 200 in A. lumbricoides. We developed a technique based on an amplification refractory mutation system (ARMS-PCR) for the analysis of 854 single A. lum bricoides eggs collected from 68 human stool samples from seven Brazilian states. We detected the mutation in codon 200 at a frequency of 0.5% (4/854). This is the first report of this mutation in A. lumbricoides. Although the observed frequency is low, its presence indi cates that these parasite populations have the potential to develop high levels of resistance in the future. The methodology proposed here provides a powerful tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations. A principal estratégia de controle para Ascaris lumbricoides é a administração em massa de medicamentos (especialmente com benzimidazóis), que podem selecionar cepas de parasitas resistentes ao tratamento. Mutações no gene isotipo-1 da beta-tubulina nos códons 167, 198 e 200 foram associados à resistência ao benzimidazólico em vários nematóides. A mutação no códon 200 é a mais frequente em diferentes espécies de parasitas, como observado anteriormente em Necator americanus e Trichuris trichiura; no entanto, esta mutação nunca foi encontrada em populações de A. lumbricoides. Este estudo teve como objetivo rastrear polimorfismos de nucleotídeo único (SNPs) na beta-tubulina gene isotype-1 no códon 200 em A. lumbricoides. Desenvolvemos uma técnica baseada em um sistema de mutação refratária à amplificação (ARMS-PCR) para a análise de 854 ovos únicos de A. lumbricoides coletados de 68 amostras de fezes humanas de sete estados brasileiros. Nós detectaram a mutação no códon 200 com uma frequência de 0,5% (4/854). Este é o primeiro relato de esta mutação em A. lumbricoides. Embora a frequência observada seja baixa, sua presença indica que essas populações parasitárias têm potencial para desenvolver altos níveis de resistência no futuro. A metodologia aqui proposta fornece uma ferramenta poderosa para a triagem de surgimento de mutações de resistência a anti-helmínticos em populações de nematóides parasitas.

8p.-3 fig.-1 tab., Clinically available drugs for mucocutaneous and cutaneous leishmaniases (CL) include mainly pentavalent antimony (Sb(V)) complexes, liposomal amphotericin B, and miltefosine (HePC). However, they present at least one of the following limitations: long-term parenteral administration through repeated doses, severe side effects, drug resistance, and high cost. HePC is the only oral drug available, but the appearance of resistance has resulted in changes of its use from monotherapy to combination therapy. Amphiphilic Sb(V) complexes, such as SbL8 obtained from reaction of Sb(V) with N-octanoyl-N-methylglucamide, were recently found to be orally active against experimental CL. The property of SbL8 to self-assemble in aqueous solution, forming nanostructures, led us to investigate the incorporation of HePC into SbL8 nanoassemblies and the therapeutic efficacy of SbL8/HePC nanoformulation by oral route in a murine model of CL. HePC incorporation into the SbL8 nanosystem was evidenced by using a fluorescent analog of HePC. The antileishmanial activity of SbL8/HePC nanoassemblies was evaluated after daily oral administration for 30 days in Leishmania amazonensis-infected BALB/c mice, in comparison with monotherapies (SbL8 or HePC) and saline control. All the treatments resulted in significant reduction in the lesion size growth, when compared with control. Strikingly, only SbL8/HePC nanoassemblies promoted a significant decrease of the parasite burden in the lesion. This work establishes the therapeutic benefit of SbL8/HePC association by oral route in a CL model and constitutes an important step towards the development of new orally active drug combination., This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 425332/2018–7), Coordenação de Aperfeicoamento de Pessoal de Nível Superior (CAPES, grant number: PNPD20131163), and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, grant number: APQ-03129-16) for financial support. F.F. was recipient of fellowship from CNPq (grant number: 305659/2017-0) and Chaire Jean d’Alembert, Université Paris-Saclay, France (ANR-11-IDEX-0003-02). L.R was supported by Program of Redes Temáticas de Investigación Cooperativa RETICS-FEDER (grant number: RD16/0027/0010).

The serodiagnosis of visceral leishmaniasis (VL) presents problems related to the sensitivity and/or specificity of the tests. In this context, more refined antigens should be identified and applied for the improvement of disease diagnosis. In the present study, DNA with an encoding of a Leishmania infantum hypothetical protein, LiHyC, was cloned, and the recombinant protein was expressed, purified, and evaluated for the serodiagnosis of canine and human VL. In addition, a specific B-cell epitope present in the LiHyC sequence was predicted; the peptide was both synthetized and evaluated in the ELISA experiments. For comparison, commercial diagnostic kits were used against positive (VL hosts) and negative (healthy hosts) samples. Results showed that the recombinant protein (rLiHyC) and synthetic peptide (PeptC) were highly sensitive and specific to diagnose canine and human VL, with 100% sensitivity and specificity, while no false-positive or false-negative result was detected. When the DPP® CVL kit was used to identify canine samples, 44 and 52 of the 60 L. infantum-infected animals, without or with clinical signals of disease, respectively, were identified, while eight and four samples were considered as false-negatives, respectively. For human VL, an IT LEISH® kit was used, and 33 of the 40 VL patients were identified, while seven samples were considered to be false-negatives. Post-therapeutic serological follow-up testing sera samples from treated and untreated VL patients showed a significant drop in the anti-PeptC and anti-rLiHyC antibody levels, thus suggesting the feasibility to use the recombinant protein and/or synthetic peptide in future studies as diagnostic and/or prognostic markers for VL.

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I2 = 90.7%; 87% CI = (84–89), P

Background Ascaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model. Methodology/Principal findings As37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules. Conclusions/Significance As37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections., Author summary Ascaris infection is the most common infection of humans living in poverty worldwide and can result in malnutrition and stunted physical and mental development in children. A preventive vaccine is urgently needed as a complementary approach to anthelmintic deworming to increase the efficiency of STH infection control. To develop a vaccine against Ascaris infection, an immunodominant antigen, As37 of A. suum, was cloned and expressed as a soluble recombinant protein in E. coli. The recombinant As37 protein (rAs37) was highly recognized by protective immune sera from A. suum infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with the adjuvant AddaVax™ showed significant larval worm reduction against challenge with A. suum infective eggs when compared to a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with a mixed Th1/2-type immune response characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. The AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 was a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, A. ceylanicum, A. caninum) and in the whipworm T. muris, but there was no cross-reaction with human spleen tissue extracts. These results indicate that the nematode-conserved As37 protein could be developed as a pan-helminth vaccine antigen to prevent all STH infections without reacting with human IgSF molecules.

Visceral leishmaniasis (VL) is a zoonosis and the dog is considered the most important urban reservoir. Cases in cats have been reported, but little is known about Leishmania infection and disease in wild felids and canids kept in captivity in endemic areas. Thus, the serological pattern of wild felids and canids kept in captivity at the Belo Horizonte Zoological Garden was investigated using two primary antigens for conventional ELISA: k39 and rKDDR, as well as three serological rapid kits: Dual Path Platform (DPP®) immunochromatographic test, rKDDR Immunochromatographic assay and ELISA SNAP Leishmania IDEXX®. A total of 21 serum samples, 13 of wild felids and 8 wild canids of varying age and sex were evaluated. The results obtained in the tests were analyzed by agreement using Kappa coefficient, and between ELISA antigens all the analysis performed had showed significant agreement among both of them, as well between the three immunochromatographic tests. The results demonstrated that there is serological evidence of wild animals seropositive for Leishmania antibodies at the Belo Horizonte Zoological Garden, and that all the antigens and rapid tests used can be employed in serological screening for VL in wild felids and canids.

The Kato-Katz (KK) technique is the mainstay mapping tool for the diagnosis of Schistosoma mansoni infection, despite showing poor sensitivity in cases of low-intensity infections. As an alternative, a rapid point-of-care circulating cathodic antigen diagnostic test (POC-CCA) has been commercially developed that involves a simple urine assay to detect S. mansoni, rather than a stool-based parasitological examination. Although POC-CCA has proven to be a more sensitive test than KK, it is not yet clear how to interpret discordant results between the two tests, particularly for situations in which the KK result is positive and the POC-CCA result is negative. Thus, the objective of this study was to evaluate the degree of diagnostic variability between different POC-CCA batches with respect to results obtained with KK. For this purpose, we collected urine and stool samples of school-aged children from areas of low and moderate endemicity in Brazil, and compared different POC-CCA batches results with those of KK-positive individuals. We found a statistically significant difference between the results obtained from various POC-CCA batches using the same urine samples, regardless of the degree of endemicity and the intensity of infection in positive KK samples. In addition, there was poor agreement between the KK and POC-CCA results in some batches of the rapid test, resulting in false negatives. These findings raise concerns around quality control checks of POC-CCA, especially in light of the high cost and increasing reliance on this new diagnostic method as control programs move towards a goal of elimination.

Background Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI-TcVI. TcII is among the major DTUs enrolled in human infections in South America southern cone, where it is associated with severe cardiac and digestive symptoms. Despite the importance of TcII in Chagas disease epidemiology and pathology, so far, no genome-wide comparisons of the mitochondrial and nuclear genomes of TcII field isolates have been performed to track the variability and evolution of this DTU in endemic regions. Results In the present work, we have sequenced and compared the whole nuclear and mitochondrial genomes of seven TcII strains isolated from chagasic patients from the central and northeastern regions of Minas Gerais, Brazil, revealing an extensive genetic variability within this DTU. A comparison of the phylogeny based on the nuclear or mitochondrial genomes revealed that the majority of branches were shared by both sequences. The subtle divergences in the branches are probably consequence of mitochondrial introgression events between TcII strains. Two T. cruzi strains isolated from patients living in the central region of Minas Gerais, S15 and S162a, were clustered in the nuclear and mitochondrial phylogeny analysis. These two strains were isolated from the other five by the Espinhaço Mountains, a geographic barrier that could have restricted the traffic of insect vectors during T. cruzi evolution in the Minas Gerais state. Finally, the presence of aneuploidies was evaluated, revealing that all seven TcII strains have a different pattern of chromosomal duplication/loss. Conclusions Analysis of genomic variability and aneuploidies suggests that there is significant genomic variability within Minas Gerais TcII strains, which could be exploited by the parasite to allow rapid selection of favorable phenotypes. Also, the aneuploidy patterns vary among T. cruzi strains and does not correlate with the nuclear phylogeny, suggesting that chromosomal duplication/loss are recent and frequent events in the parasite evolution. Electronic supplementary material The online version of this article (10.1186/s12864-018-5198-4) contains supplementary material, which is available to authorized users.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

Summary We have recently developed a sensitive and specific urine‐based antigen detection ELISA for the diagnosis of visceral leishmaniasis (VL). This assay used rabbit IgG and chicken IgY polyclonal antibodies specific for the Leishmania infantum proteins iron superoxide dismutase 1 (Li‐isd1), tryparedoxin1 (Li‐txn1) and nuclear transport factor 2 (Li‐ntf2). However, polyclonal antibodies have limitations for upscaling and continuous supply. To circumvent these hurdles, we began to develop immortalized monoclonal antibodies. We opted for recombinant camelid VHHs because the technology for their production is well established and they do not have Fc, hence providing less ELISA background noise. We report here an assay development using VHHs specific for Li‐isd1 and Li‐ntf2. This new assay was specific and had analytical sensitivity of 15‐45 pg/mL of urine. The clinical sensitivity was comparable to that obtained with the ELISA assembled with conventional rabbit and chicken antibodies to detect these two antigens. Therefore, similar to our former studies with conventional antibodies, the future inclusion of VHH specific for Li‐txn1 and/or other antigens should further increase the sensitivity of the assay. These results confirm that immortalized VHHs can replace conventional antibodies for the development of an accurate and reproducible antigen detection diagnostic test for VL.

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity.

In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the serodiagnosis of canine and human leishmaniasis.

Objectives To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. Methods Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. Results Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. Conclusions Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens. Objectifs Identifier des antigenes immunodominants de Toxocara canis reconnus par des serums infectes par Toxocara, comme recombinants reactifs pour le diagnostic immunologique de la toxocarose. Methodes Des serums pooles provenant de cas humains de toxocarose ont ete utilises pour identifier des antigenes immunodominants par immunocriblage d'une banque d'expression d’ADNc de larves de T. canis. Les clones positifs ont ete sequences pour reveler l'identite des antigenes. Les proteines recombinantes ont ete exprimees dans E. coli et ensuite utilisees pour confirmer leur reaction immunologique avec des serums humains de toxocarose. Deux antigenes ont egalement ete utilises pour differencier la toxocarose d'autres helminthiases chez la souris. Resultats 11 antigenes a potentiel d'immunodiagnostic ont ete identifies, dont deux lectines de type C (CTL) qui ont reagi fortement avec le pool de serums positifs pour Toxocara. La premiere CTL (Tc-CTL-1) est la meme que TES-32, precedemment identifiee comme une composante immunodominante majeure de TES; la deuxieme CTL (Tc-CTL-2) est une nouvelle lectine de type C partageant une identite de 83% avec la sequence d'acides amines dans la region fonctionnelle de Tc-CTL-1. Le recombinant Tc-CTL-1 exprime dans E. coli a ete fortement reconnu par le pool de serums positifs pour Toxocara ou des serums provenant d'animaux infectes experimentalement avec T. canis. La reactivite avec le recombinant Tc-CTL-1 etait plus forte lorsque la proteine non reduite a ete utilisee dans des essais ELISA, Dot-blot ou Western-blot par rapport a la proteine sous sa forme reduite. Les ELISA bases sur les deux recombinants Tc-CTL-1 et Tc-CTL-2 ont ete en mesure de differencier l'infection a T. canis d'autres helminthiases chez la souris infectee experimentalement. Conclusions Tc-CTL-1 et Tc-CTL-2 ont ete en mesure de differencier la toxocarose d'autres helminthiases et pourraient potentiellement etre utilises comme antigenes d'immunodiagnostic sensibles et specifiques. Objetivos Identificar antigenos inmunodominantes de Toxocara canis reconocidos por sueros infectados con Toxocara como reactivos recombinantes para el inmunodiagnostico de la toxocariasis. Metodos Se utilizo un pool de sueros de casos de toxocariasis humana para identificar los antigenos inmunodominantes mediante inmunoscreening en una biblioteca de ADNc de larvas de T. canis. Los clones que dieron positivo fueron secuenciados para identificar los antigenos. Las proteinas recombinantes se expresaron en E. coli y se utilizaron para confirmar su inmunoreactividad con sueros de humanos con toxocariasis. Dos antigenos especificos se utilizaron tambien para diferenciar la infeccion por Toxocara de otras infecciones por helmintos en raton. Resultados Se identificaron 11 antigenos con potencial inmunodiagnostico, incluyendo dos lectinas del tipo C (CLT) que reaccionaban fuertemente con el pool de sueros Toxocara-positivos. La primera LTC (Tc- CLT -1) es la misma que TES-32, previamente identificada como el principal componente inmunodominante de TES; la segunda LTC (Tc- CLT -2) es una nueva lectina tipo C que comparte un 83% de la secuencia de aminoacidos dentro del dominio funcional de Tc- CLT -1. La Tc- CLT -1 recombinante expresada en E. coli era altamente reconocida por el pool de sueros Toxocara-positivos o sueros de animales infectados experimentalmente con T. canis. La reactividad con el Tc- CLT -1 recombinante era mayor cuando se utilizaba la proteina bajo condiciones no reductoras en un ELISA, Dot blot o Western blot comparado con la proteina en condiciones reductoras. Las ELISAs en las que se utilizaban la Tc- CLT -1 recombinante y la Tc- CLT -2 recombinante eran capaces de diferenciar la infeccion por T. canis de otras infecciones helminticas en ratones infectados experimentalmente. Conclusiones Tanto Tc- CLT-1 como Tc- CLT-2 eran capaces de diferenciar la infeccion por Toxocara de otras infecciones helminticas y potencialmente podrian utilizarse como antigenos sensibles y especificos para inmunodiagnostico.

Toxic effects of Peruvian Hadruroides lunatus scorpion venom on different biochemical and enzymatic parameters in blood serum of Wistar rats and Swiss mice were determined after experimental envenomation. An increase in enzymatic activities of Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH) and levels of serum protein and albumin were observed while a decrease in creatinine level in serum was perceived after 30 min of envenomation. No alterations in urea levels and in kidney histology were detected in the envenomed rats. The global leukocytes count was diminished, with decrease in lymphocytes, eosinophils and neutrophils levels in the bloodstream, while no alterations were found in hematological parameters of red series in rats injected with H. lunatus venom. IL-2, IL-4, IL-6, INF-γ, TNF, IL-17A and IL-10 levels were evaluated 0.5, 3 and 6 h after experimental envenomation of mice with H. lunatus venom. From all the analyzed cytokines, only IL-6 showed an increase in serum levels. Taken together, these results point out that envenomation by H. lunatus can impair hematological and immunological parameters and therefore might be monitored in accidents involving this species.

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions.

Live attenuated Leishmania donovani parasites such as LdCen(-/-) have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen(-/-) including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4(+) and CD8(+) T cells, IFN-γ production by CD8(+) T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen(-/-) parasites as vaccine candidates, we performed a 24-month follow up of LdCen(-/-) immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen(-/-) (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune(®)). The protection was associated with antibody production and CD4(+) and CD8(+) proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune(®), with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen(-/-) expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune(®) or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.