RESUMO
Trypanosoma cruzi, the etiologic agent of American trypanosomiasis, is a vector-borne zoonotic parasite which has been little studied regarding its infection in domestic animals. In this study, we evaluated the occurrence of natural infection by T. cruzi in farm animals using molecular markers and phylogenetic analysis in blood clot samples of 60 sheep (Ovis aires), 22 goats (Capra hircus), and 14 horses (Equus caballus) in eight municipalities located in an infection risk area in the state of Rio Grande do Norte (RN), Northeast Region of Brazil. Trypanosoma spp. infection was identified by amplifying the rRNA 18S SSU gene in 48.9% of the samples. The SH022 sample showed 99.8% similarity with the Y strain of T. cruzi in phylogeny, grouped in the DTU II clade. Blood clots of sheep, goats, and horses detected T. cruzi kDNA in 28.3% (17/60), 22.7% (5/22), and 15.4% (2/14) of the samples, respectively. These animals were distributed in the three studied mesoregions throughout the state of RN. The identification of natural infection in domestic animals contributes to expand the epidemiological transmission scenario in an area where T. brasiliensis is the main vector.
Assuntos
Doença de Chagas , Triatoma , Trypanosoma cruzi , Animais , Ovinos , Trypanosoma cruzi/genética , Animais Domésticos/parasitologia , Brasil/epidemiologia , Filogenia , Cidades , Insetos Vetores/parasitologia , Doença de Chagas/epidemiologia , Doença de Chagas/veterinária , Doença de Chagas/parasitologia , Cabras , Triatoma/genéticaRESUMO
The genetic variability of 24 Trypanosoma cruzi isolates from humans (11) and triatomines (13) in northeastern Brazil was analyzed by random amplified polymorphic DNA (RAPD) and compared with taxonomic groups, host, and geographical origin of the parasite. TcI (12.5%), TcII (45.8%), and TcIII (41.6%) showed a similarity coefficient (SC) of 0.74 using the mean of three primers and 0.80, 0.75, and 0.66 for λgt11-F, M13-40F, and L15996 primers, respectively. The samples were clustered according to their phylogenetic origin in two polymorphic and divergent branches: one associated with TcI and the other with two subbranches corresponding to TcII and TcIII. TcI was only identified in humans and correlated with the Id homogenous group (0.80 SC). TcII from humans and Triatoma brasiliensis showed 0.86 SC and was clustered according monoclonal or polyclonal populations with similar RAPD profiles detected among the vector and/or humans in different municipalities. TcIII was isolated exclusively in sylvatic cycles from T. brasiliensis and Panstrongylus lutzi and showed low variability (0.84 SC) and high homology mainly among isolated populations at the same locality. The homology of T. cruzi among different hosts and locations suggests the distribution of principal clones circulating and reveals an overlapping between the sylvatic and domestic cycles in this area, where T. brasiliensis infected with TcII acts as link in both environments. This species is important to maintain TcII and TcIII in wild cycles and deserves particular attention due an emergent risk of these populations being introduced into the domestic cycle; moreover, its clinical and epidemiological implications remain unknown.
Assuntos
Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Vetores de Doenças , Variação Genética , Triatoma/parasitologia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Animais , Brasil , Análise por Conglomerados , Impressões Digitais de DNA , DNA de Protozoário/genética , Humanos , Filogeografia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
BACKGROUND: Components of the antioxidant defense system in Trypanosoma cruzi are potential targets for new drug development. Superoxide dismutases (SODs) constitute key components of antioxidant defense systems, removing excess superoxide anions by converting them into oxygen and hydrogen peroxide. The main goal of the present study was to investigate the genes coding for iron superoxide dismutase (FeSOD) in T. cruzi strains from an evolutionary perspective. METHODS: In this study, molecular biology methods and phylogenetic studies were combined with drug assays. The FeSOD-A and FeSOD-B genes of 35 T. cruzi strains, belonging to six discrete typing units (Tcl-TcVI), from different hosts and geographical regions were amplified by PCR and sequenced using the Sanger method. Evolutionary trees were reconstructed based on Bayesian inference and maximum likelihood methods. Drugs that potentially interacted with T. cruzi FeSODs were identified and tested against the parasites. RESULTS: Our results suggest that T. cruzi FeSOD types are members of distinct families. Gene copies of FeSOD-A (n = 2), FeSOD-B (n = 4) and FeSOD-C (n = 4) were identified in the genome of the T. cruzi reference clone CL Brener. Phylogenetic inference supported the presence of two functional variants of each FeSOD type across the T. cruzi strains. Phylogenetic trees revealed a monophyletic group of FeSOD genes of T. cruzi TcIV strains in both distinct genes. Altogether, our results support the hypothesis that gene duplication followed by divergence shaped the evolution of T. cruzi FeSODs. Two drugs, mangafodipir and polaprezinc, that potentially interact with T. cruzi FeSODs were identified and tested in vitro against amastigotes and trypomastigotes: mangafodipir had a low trypanocidal effect and polaprezinc was inactive. CONCLUSIONS: Our study contributes to a better understanding of the molecular biodiversity of T. cruzi FeSODs. Herein we provide a successful approach to the study of gene/protein families as potential drug targets.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Antioxidantes , Teorema de Bayes , Doença de Chagas/parasitologia , Humanos , Filogenia , Superóxido Dismutase/genética , Superóxidos , Trypanosoma cruzi/genéticaRESUMO
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 (Qß-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.
Assuntos
Cardiomiopatia Chagásica/prevenção & controle , Coração/parasitologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi , Animais , Anticorpos Antiprotozoários/sangue , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Imunoglobulina G/sangue , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Parasitemia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Vector transmission of Trypanosoma cruzi occurs in several areas of Brazil, including the northeastern region, and domestic animals can serve as reservoirs of the parasite. The aim of this study was to monitor dogs as domestic reservoirs for infection by T. cruzi, and the main triatomine species involved in parasite transmission in rural areas of municipalities in the State of Rio Grande do Norte, in northeastern, Brazil. Blood samples from dogs (n = 40) and manual triatomine capture were performed in domiciliary and peridomiciliary environments in rural areas of the towns of Acari, Caraúbas and Marcelino Vieira, between 2013 and 2016. Subsequently, infection of dogs was determined by Polymerase Chain Reaction (PCR), Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IgM and IgG isotypes and Indirect Immunofluorescence (IIF) reactions for detection of IgG. Triatomine infection was determined by PCR. Forty (16/40) percent of the dogs were seropositive for T. cruzi; 20.0% (8/40) of such reactivity indicated the acute phase, and 20.0% (8/40), the chronic phase. PCR was positive in 42.5% (17/40) of the dogs' blood samples. Specimens of Triatoma brasiliensis, Triatoma pseudomaculata, Rhodnius nasutus and Panstrongylus lutzi were found to be infected; however only T. brasiliensis nymphs and adults were infected in both environments. Triatomines evaluation showed 82.5% (94/114) of PCR positivity. Taken together, our results confirm that dogs are domestic reservoirs of T. cruzi in northeastern Brazil and T. brasiliensis is the main triatomine species correlated with parasite transmission in domiciliary environments. There is a continuing need to control peridomiciliary populations of triatomines and to implement continuous surveillance strategies for reservoirs with the help from the community.
Assuntos
Doença de Chagas/transmissão , Cães/parasitologia , Insetos Vetores/parasitologia , Panstrongylus/parasitologia , Rhodnius/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi/isolamento & purificação , Animais , Brasil/epidemiologia , Doença de Chagas/epidemiologia , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Ninfa/genética , Reação em Cadeia da Polimerase , Trypanosoma cruzi/genéticaRESUMO
The protozoan Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that afflicts approximately 8 million people in Latin America. Diagnosis of chronic Chagas disease is currently based on serological tests because this condition is usually characterized by high anti-T. cruzi IgG titers and low parasitemia. The antigens used in these assays may have low specificity due to cross reactivity with antigens from related parasite infections, such as leishmaniasis, and low sensitivity caused by the high polymorphism among T. cruzi strains. Therefore, the identification of new T. cruzi-specific antigens that are conserved among the various parasite discrete typing units (DTUs) is still required. In the present study, we have explored the hybrid nature of the T. cruzi CL Brener strain using a broad genome screening approach to select new T. cruzi antigens that are conserved among the different parasite DTUs and that are absent in other trypanosomatid species. Peptide arrays containing the conserved antigens with the highest epitope prediction scores were synthesized, and the reactivity of the peptides were tested by immunoblot using sera from C57BL/6 mice chronically infected with T. cruzi strains from the TcI, TcII or TcVI DTU. The two T. cruzi proteins that contained the most promising peptides were expressed as recombinant proteins and tested in ELISA experiments with sera from chagasic patients with distinct clinical manifestations: those infected with T. cruzi from different DTUs and those with cutaneous or visceral leishmaniasis. These proteins, named rTc_11623.20 and rTc_N_10421.310, exhibited 94.83 and 89.66% sensitivity, 98.2 and 94.6% specificity, respectively, and a pool of these 2 proteins exhibited 96.55% sensitivity and 98.18% specificity. This work led to the identification of two new antigens with great potential application in the diagnosis of chronic Chagas disease.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/química , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reprodutibilidade dos TestesRESUMO
The clinical course of Chagas' disease varies widely among different patients and geographic regions. For reasons that are not completely understood but involve host and parasite factors, some patients never develop the disease while others present cardiac and/or gastrointestinal symptoms. Many studies have been conducted in order to correlate the genetic variability of the parasites with the clinical forms of the disease, but no conclusive data have been obtained. Our research aims at characterizing the genetic profiles of Trypanosoma cruzi isolates recently obtained from 70 chagasic patients who either showed pathological lesions with symptoms of various intensities or were asymptomatic. All patients came from an area where Chagas' disease is endemic in southeast Brazil where vectorial transmission has been controlled and different clinical forms of the disease can be found. The molecular characterization of parasites evaluated the polymorphisms of the 3' region of the 24Salpha rRNA gene and the variability of kinetoplast DNA (kDNA) minicircles of T. cruzi populations by low-stringency single specific primer PCR. Data presented here provide a strong correlation between T. cruzi II and human infection in this region. However, a high degree of variability was observed within T. cruzi II, as demonstrated by intense kDNA polymorphism among all clinical forms and also within each of them, irrespective of the intensity of pathological processes.