Geographical distribution of Trypanosoma cruzi in triatomine vectors in the State of Mato Grosso do Sul, Brazil

Introduction This work presents the initial findings of a molecular epidemiological investigation of Trypanosoma cruzi in triatomine insects in State of Mato Grosso do Sul. Methods A total of 511 triatomines from different regions of the state were examined. Deoxyribonucleic acid (DNA) was extracted from the intestinal contents of the insects using phenol-chloroform-isoamyl alcohol (25:24:1). Polymerase chain reaction (PCR) using primers 121/122 targeting DNA kinetoplast (kDNA) was then performed to identify T. cruzi, and positive samples were subjected to PCR using the primer pair TcSC5D-F/R followed by restriction fragment length polymorphism (RFLP) with the restriction enzymes SphI and HpaI (1 U/reaction), cloning and sequencing. Results One hundred samples were positive for T. cruzi, and three discrete typing units (DTUs) were identified (TcI, TcII, and TcBat). Triatoma sordida had the highest T. cruzi occurrence (83.3%), and DTUs were found in three samples: 58.3% of the samples were TcI, 33.3% were TcII and 8.3% were TcBat. There was a clear geographical distribution of the DTUs throughout the state, with TcI, TcII and TcBat located in the center, TcI located in the east, and TcII located in the west. Conclusions This study showed the occurrence of overlapping DTUs in State of Mato Grosso do Sul. The distributions of the DTUs were different, with TcI, TcII and TcBat in the center of the state, TcI predominantly in the east, and TcII in the west. Further studies may reveal a more defined mosaic distribution of DTUs in MS.

Detection of immunogenic proteins from Anopheles sundaicussalivary glands in the human serum

AbstractINTRODUCTION:The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specific IgG antibodies in the host, which can be used as specific biomarkers of exposure to Anopheles sundaicus . The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes.METHODS:IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting.RESULTS:Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls.CONCLUSIONS:These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines.

Triatominae (Hemiptera, Reduviidae) in the Pantanal region: association with Trypanosoma cruzi, different habitats and vertebrate hosts

ABSTRACTINTRODUCTION: The transmission cycle of Trypanosoma cruzi in the Brazilian Pantanal region has been studied during the last decade. Although considerable knowledge is available regarding the mammalian hosts infected by T. cruzi in this wetland, no studies have investigated its vectors in this region. This study aimed to investigate the presence of sylvatic triatomine species in different habitats of the Brazilian Pantanal region and to correlate their presence with the occurrences of vertebrate hosts and T. cruzi infection.METHODS: The fieldwork involved passive search by using light traps and Noireau traps and active search by visual inspection. The light traps were placed at five selected points along forested areas for seven nights during each of the nine excursions. At each point where a light trap was set, eight Noireau traps were placed in palm trees and bromeliads.RESULTS: In all, 88 triatomine bugs were collected: two and one individuals from light traps and Noireau traps, respectively; three from peridomestic areas; 23 in coati nests; and 59 in thornbird nests. In this study, active search in microhabitats showed higher efficiency than passive search, since 95% of the triatomine bugs were caught in nests. Further, triatomine bugs were only found to be infected by T. cruzi in coati nests.CONCLUSIONS: Coati nests might act as a point of convergence and dispersion for triatomine bugs and mammal hosts infected by T. cruzi, thereby playing an important role in the sylvatic cycle of T. cruziin the Pantanal region.

Anopheles darlingi and Anopheles marajoara (Diptera: Culicidae) susceptibility to pyrethroids in an endemic area of the Brazilian Amazon

Abstract: INTRODUCTION: This study aimed to evaluate the susceptibility of Anopheles darlingi Root (1926) and Anopheles marajoara Galvão & Damasceno (1942) to pyrethroids used by the National Malaria Control Program in Brazil. METHODS: Mosquitoes from Amapá, Brazilian Amazon, were assessed for resistance to cypermethrin, deltamethrin, and alpha-cypermethrin. Insecticide-impregnated bottles were used as suggested by the CDC/Atlanta. RESULTS: Diagnostic dose for Anopheles darlingi was 12.5µg/bottle during 30 min of exposure. Concentrations for Anopheles marajoara were 20µg/bottle of cypermethrin and deltamethrin and 12.5µg/bottle of alpha-cypermethrin. CONCLUSIONS : No resistance was recorded for Anopheles darlingi , but Anopheles marajoara requires attention.

Characteristics of Triatomine infestation and natural Trypanosoma cruzi infection in the State of Rio Grande do Norte, Brazil

Abstract: INTRODUCTION Natural and artificial ecotope infestation by the kissing bug triatomines and their colonization and infection by Trypanosoma cruzi , the Chagas disease agent, were evaluated in nine municipalities of the State of Rio Grande do Norte, Brazil. METHODS Following identification, triatomine intestinal contents were analyzed by direct microscopic examination, xenoculture, and polymerase chain reaction (PCR) for parasite detection. Trypanosoma cruzi isolates were genotyped using three different markers. RESULTS Of 842 triatomines captured, 65% were Triatoma brasiliensis , 17.8% Triatoma pseudomaculata , 12.5% Panstrongylus lutzi , and 4.7% Rhodnius nasutus . Triatoma brasiliensis and P. lutzi adults were found in the intradomicile. T. brasiliensis, T. pseudomaculata , and R. nasutus nymphs and adults were found in the peridomicile and wild environment. Intradomiciliary and peridomiciliary infestation indexes were 5.6% and 33.7%, respectively. In the peridomicile, chicken coops were the most infested ecotope. The T. cruzi triatomine infection rate was 30.2%, of which PCR detected 29%. P . lutzi (78.1%), T . brasiliensis (24.5%), and T . pseudomaculata (22.7%) were the most infected species. TcII and III genotypes were detected in T. brasiliensis and TcIII in P. lutzi . CONCLUSIONS T. brasiliensis was found in all environments and most ecotopes with high T. cruzi infection rates. High infection rates were also detected in T . pseudomaculata and P. lutzi , suggesting their role in the interchange between the wild and peridomestic transmission cycles. The combination of PCR, microscopic examination, and xenoculture contributed to improving T. cruzi infection evaluation in triatomine bugs. The TcII and TcIII genotypes were predominant in the study area.

Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America

Abstract: INTRODUCTION: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. METHODS: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. RESULTS: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. CONCLUSIONS: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

Extraction of Trypanosoma cruzi DNA from food: a contribution to the elucidation of acute Chagas disease outbreaks

Abstract: INTRODUCTION: Before 2004, the occurrence of acute Chagas disease (ACD) by oral transmission associated with food was scarcely known or investigated. Originally sporadic and circumstantial, ACD occurrences have now become frequent in the Amazon region, with recently related outbreaks spreading to several Brazilian states. These cases are associated with the consumption of açai juice by waste reservoir animals or insect vectors infected with Trypanosoma cruzi in endemic areas. Although guidelines for processing the fruit to minimize contamination through microorganisms and parasites exist, açai-based products must be assessed for quality, for which the demand for appropriate methodologies must be met. METHODS: Dilutions ranging from 5 to 1,000 T. cruzi CL Brener cells were mixed with 2mL of acai juice. Four Extraction of T. cruzi DNA methods were used on the fruit, and the cetyltrimethyl ammonium bromide (CTAB) method was selected according to JRC, 2005. RESULTS: DNA extraction by the CTAB method yielded satisfactory results with regard to purity and concentration for use in PCR. Overall, the methods employed proved that not only extraction efficiency but also high sensitivity in amplification was important. CONCLUSIONS: The method for T. cruzi detection in food is a powerful tool in the epidemiological investigation of outbreaks as it turns epidemiological evidence into supporting data that serve to confirm T. cruzi infection in the foods. It also facilitates food quality control and assessment of good manufacturing practices involving acai-based products.

Life cycle of Trypanosoma cruzi (y strain) in mice

Since 1958, we have studied experimental Chagas' disease (CD) by subcutaneous inoculation of 1,000 blood forms of Trypanosoma cruzi (Y strain) in Balb/C. mice. Evolution of parasitemia remained constant, beginning on the 5th and 6th day of the disease, increasing progressively, achieving a maximum on about the 30th day. After another month, only a few forms were present, and they disappeared from the circulation after the third month, as determined from direct examination of slides and the use of a Neubauer Counting Chamber. These events coincided with the appearance of amastigote nests in the tissues (especially the cardiac ones), starting the first week, and following the Gauss parasitemia curve, but they were not in parallel until the chronic stage. In 1997, we began to note the following changes: Parasites appeared in the circulation during the first week and disappeared starting on the 7th day, and there was a coincident absence of the amastigote nests in the tissues. A careful study verified that young forms in the evolutionary cycle of T. cruzi (epi + amastigotes) began to appear alongside the trypomastigotes in the circulation on the 5th and 7th post-inoculation day. At the same time, rounded, oval, and spindle shapes were seen circulating through the capillaries and sinusoids of the tissues, principally of the hematopoietic organs. Stasis occurs because the diameter of the circulating parasites is greater than the vessels, and this makes them more visible. Examination of the sternal bone marrow revealed young cells with elongated forms and others truncated in the shape of a "C" occupying the internal surface of the blood cells that had empty central portions (erythrocytes?). We hypothesize that there could be a loss of virulence or mutation of the Y strain of Trypanosoma cruzi.

Biologia de Anofelinos Amazônicos. VI. Variabilidade Enzimática em ANOPHELES DARLINGI Root. (DIPTERA, CULICIDAE)

São relatados resultados preliminares sobre o polimorfismo protético de populuções naturais de Anopheles darlingi, procedentes de duas localidades da região amazônica-Ariquemes (Rondônia) e Rodovia PA-422 (Pará). Pata as esterases, foi detectada variação para o locus Est-2na população de Ariquemes, enquanto foi monomórfico na população da PA-422, sendo verificados nesta última, apenas indivíduos com o genótipo Est2*F/Est2*F. A freqüência do alelo Est2*F foi estimada em 0,80 na população de Ariquemes, valor este muito maior do que o observado na população da BR-174 (Manaus/Boa Vista), em estudos anteriores, que foi de 0,48. Os resultados para o sistema da leucina aminopeptidase (LAP) sugerem que a enzima é controlada por três loci, aparentemente monomórficos em ambas as populações.

Biology of amazonian mosquitoes. III. Esterase isozymes in Anopheles darlingi.

Six esterase isozymes were studied during the development of Anopheles darlingi by using polyacrylamide gel electrophoresis and two different substrates, a-naphthylcelate and a-naphthylpropionate. Esterases 5 and 6'were detected in all developmental stages esterases 1 and 2 were more intensively stained if larvae, while esterases 3 and 4 were better visualized in pupae and adults. Strong differences in intensity of some of the isozymes were observed during the pupal stage.Four out of the six isozymes showed variation in the electrophoretic mobility. Esterase-2 was choosed for genetic studies, because was the best stained isozyme in the gels. Two codominant alleles {Est2*S and Est2*F) code for this polymorphic system, with the Est*S frequency equal to 0.521. Phenotypic distribution is in agreement with hardy-Weinberg expectations.

Biologia de anofelinos Amazônicos. XI. Estudos em populações de Anopheles e controle da malária em Ariquemes (Rondônia)

São apresentados dados sobre inquérito entomológico para espécies de Anopheles rea lizado no Município de Ariquemes e discutida a incidência da malária neste Município e no Estado de Rondônia. Os resultados do inquérito entomológico na zona urbana de Ariquemes evidenciaram a diversidade de espécies nos diferentes Setores, possibilitando verificar o grau de penetração dos anofelinos na cidade. São correlacionados casos autóctones de malária e a presença do principal vetor da malária humana na Amazônia, o Anopheles darlingi. A dinâmica da transmissão da malária na zona urbana é enfocada, discutindo-se os riscos verificados para cada Setor e são apresentadas estratégias de controle, ao nível do vetor, fundamentadas no manuseio do habitat. Να zona rural estão sendo estudados parâmetros populacionais relacionados (1) à atividade de picar, abrigo natural, sí-tios de reprodução e deslocamento para hematofagia; e (2) comportamento das populações para atividade hematófaga e preferências alimentares. Os resultados, embora preliminares, evidenciam variações na densidade populacional e na diversidade de espécies, as quais devem estar relacionadas à mudanças estacionais. Variações nas preferências alimentares das espécies também foram verificadas, sendo constatada elevada antropofilia de A. darlingi. Dois parâmetros relevantes na atividade hematófaga de A. darlingi foram detectados, observando se que os anofelinos estão penetrando nas habitações petas partes inferiores e que não estão pousando nas paredes com DDΤ. Na vegetação circundante às habitações, está havendo preferência para locais de pouso, que podem ser alternativamente alto e baixo.

Estudo de mecanismos envolvidos na incorporação de proteínas durante a ovogênese em Anopheles

Ovários de Anopheles triannulatus foram dissecados 24 e 48 horas após as fêmeas terem sido alimentadas com fonte protéica (sangue), afim de se estudar o seu efeito sobre a ovogênese. Foram analisadas secçõees hisiológicas, ao nível da ultraestrutura, e observadas as mudanças morfológicas que ocorrem nas interfaces células foliculares-ovócito e célula folicular-célula. Foram observados aspectos estruturais que corroboram a hipótese da produção exógena das proteínas do vitelo. Os grânulos de vitelo originaram-se principalmente por condensação das vesículas pinocíticas formadas na superfície do ovócito.

Biologia de Anofelinos Amazônicos. XIII. Estudo do ciclo biológico de Anopheles nuñez-tovari Gabaldon, 1940 (Diptera, Culicidae)

No estudo do ciclo biológico de Anopheles nuñez-tovari foram analisadas 108 posturas, totalizando 13.999 ovos. A média de ovos postos por postura foi de 128,83 + 4,85 e uma taxa de eclosão elevada de 84,06%. Foi constatado qe 89% dos ovos eclodem com 48 horas após a oviposição, mostrando, no total, um período médio de eclosão de 1,86 + 0,004 dias. A média de ovos eclodidos por postura foi de 109,39 + 5,26. Os dados de correlação entre ovos postos e ovos eclodidos evidenciaram que à medida que aumenta o número de ovos por postura há também um aumento correspondente na taxa de eclosão . Contudo, quando as posturas são reunidas em intervalos de classe de 40 ovos, constatou-se que não há correlação para algumas delas. Para as fases imaturas, verificou-se que existem diferenças no tempo de desenvolvimento, sendo o estádio 4o - pupa - o mais longo, apresentando uma média de 3,15 + 0,03 dias. A menor média foi observada para os estádios 2o - 3o com 1,46 + 0,02 dias. Valores intermediários foram constatados para os estádios 1o - 2o, 3o - 4oe estágio de pupa - adulto (2,34 + 0,02; 1,67 + 0,02 e 1,54 + 0,01 dias, respetivamente). Para o ciclo completo ovo-adulto observou-se um tempo médio de desenvolvimento de 12,02 + 0,04 dias. As maiores taxas de mortalidade foram verificadas para o 4o. estádio (19,6%0) e para o estádio até a emergência do imago, foi de 71,40%, sendo um valor elevado segundo os dados da literatura. A comparação dos períodos do desenvolvimento entre A. nuñez-tovari com A. darlingi e A. trannulatuspossibilitou considerar A. nuñez-tovari como mais próxima da primeira espécie.

BIOLOGIA DE ANOFELINOS AMAZÔNICOS. XIV. ISOENZIMAS DE ESTERASE EM ANOPHELES TRIANNULATUS (NEIVA & PINTO, 1922)

Foram estudadas populações de Anopheles triannulatusprocedentes da hidrelétrica de Balbina (AM), quanto ao sistema das esterases, usando-se gel de poliacrilamida e como substrato α-naftil propionato A análise eletroforética possibilitou identificar seis zonas de atividade e o mecanismo de herança foi estudado para cinco locique foram polimórficos. Foram detectados dois alelos para as esterases 1, 2 e 4 e três para as esterases 3 e 5, sendo todos de ação codominante. Os dados de frequências gênicas mostraram-se diferentes para os alelos em cada locus.Os valores de qui-quadrados para as frequências genotípicas evidenciaram que a população não está em equilíbrio para todos os loci,sendo as frequências observadas dos homozigotos superiores as esperadas. Esses resultados foram interpretados como decorrentes de alterações ambientais que estão ocorrendo na área em estudo.

BIOLOGIA DE ANOFELINOS AMAZÔNICOS. XV. LEUCINA AMINOPEPTIDASE EM ANOPHELES (NYSSORHYNCHUS) NUÑEZ-TOVARI; ONTOGENIA E VARIAÇÃO GENÉTICA

Dados sobre a leucina aminopeptidase durante o desenvolvimento ontogenético em Anopheles nuñez-tovarìevidenciaram seis zonae de atividade, eendo a LAP1, LAP2, LAP4 e LAP5 presentes nos estádios larvais, pupae e adultos e a LAP3 e LAP6 características desses dois últimos estágios. Diferenças na intensidade de coloração foram detectadas conforme o estágio considerado. A LAP1 e LAP5 apresentam intensidade fraca em todos os estágios e a LAP2 e LAP4 mostram atividade intensa nos estádios larvais, diminuindo nos dois estágios subseqüentes. Considerando-se as funções das enzimas, foi admitido que a LAP3, no estágio de pupa, possa estar relacionada com a histólise dos tecidos larvais. Dos seis locidetectados, variação alélica foi constatada apenas para o locus LAP5,sendo detectados dois alelos de ação codominante. As freqüências genotípicas de progênies de fêmeas inseminadas naturalmente desviam-se significativamente do equilíbrio de Hardy-Weinberg.