Archaea Symbiont of T. cruzi Infection May Explain Heart Failure in Chagas Disease.

Background: Archaeal genes present in Trypanosoma cruzi may represent symbionts that would explain development of heart failure in 30% of Chagas disease patients. Extracellular vesicles in peripheral blood, called exosomes (< 0.1 µm) or microvesicles (>0.1 µm), present in larger numbers in heart failure, were analyzed to determine whether they are derived from archaea in heart failure Chagas disease. Methods: Exosomes and microvesicles in serum supernatant from 3 groups were analyzed: heart failure Chagas disease (N = 26), asymptomatic indeterminate form (N = 21) and healthy non-chagasic control (N = 16). Samples were quantified with transmission electron microscopy, flow cytometer immunolabeled with anti-archaemetzincin-1 antibody (AMZ 1, archaea collagenase) and probe anti-archaeal DNA and zymography to determine AMZ1 (Archaeal metalloproteinase) activity. Results: Indeterminate form patients had higher median numbers of exosomes/case vs. heart failure patients (58.5 vs. 25.5, P < 0.001), higher exosome content of AMZ1 antigens (2.0 vs. 0.0; P < 0.001), and lower archaeal DNA content (0.2 vs. 1.5, P = 0.02). A positive correlation between exosomes and AMZ1 content was seen in indeterminate form (r = 0.5, P < 0.001), but not in heart failure patients (r = 0.002, P = 0.98). Higher free archaeal DNA (63.0 vs. 11.1, P < 0.001) in correlation with exosome numbers (r = 0.66, P = 0.01) was seen in heart failure but not in indeterminate form (r = 0.29, P = 0.10). Flow cytometer showed higher numbers of AMZ1 microvesicles in indeterminate form (64 vs. 36, P = 0.02) and higher archaeal DNA microvesicles in heart failure (8.1 vs. 0.9, P < 0.001). Zymography showed strong% collagenase activity in HF group, mild activity in IF compared to non-chagasic healthy group (121 ± 14, 106 ± 13 and 100; P < 0.001). Conclusions: Numerous exosomes, possibly removing and degrading abnormal AMZ1 collagenase, are associated with indeterminate form. Archaeal microvesicles and their exosomes, possibly associated with release of archaeal AMZ1 in heart failure, are future candidates of heart failure biomarkers if confirmed in larger series, and the therapeutic focus in the treatment of Chagas disease.

Posible transmisión oral de la enfermedad de Chagas en trabajadores del sector de los hidrocarburos en Casanare, Colombia, 2014 / Possible oral transmission of Chagas disease among hydrocarbons sector workers in Casanare, Colombia, 2014

RESUMEN Introducción. Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, puede transmitirse por vía oral tras la ingestión de alimentos o bebidas contaminadas. En la semana epidemiológica 14 del 2014, se notificaron dos casos de enfermedad aguda de Chagas en Paz de Ariporo, Casanare, entre trabajadores del sector de los hidrocarburos, episodio que motivó la investigación epidemiológica en el área. Objetivo. Caracterizar la población afectada, establecer medidas de control y confirmar el brote. Materiales y métodos. Se hizo una investigación de brote con los siguientes componentes: a) búsqueda de personas sintomáticas (cuadro clínico sugerente de enfermedad de Chagas según la definición de caso), para remitirlas a atención médica; b) aplicación de una encuesta entomológica en 192 de 197 viviendas; c) inspección sanitaria y análisis microbiológico de muestras de alimentos, y d) estudio de reservorios. La organización y el análisis de los datos se hicieron mediante estadística descriptiva con el programa Epi-Info 7.1.5. Asimismo, se establecieron los índices de infestación en el domicilio y el peridomicilio. Resultados. Se registraron 552 personas expuestas y se confirmaron por laboratorio 40 casos (7,2 %); siete casos se dieron en mujeres (17,5 %) y 33 en hombres (82,5%), es decir, en una relación de 1:5. La edad promedio fue de 39,1 (± 10,8) años, la tasa de ataque, de 7,2 %, y la letalidad, de 5 % (2/40). Los signos y síntomas incluyeron fiebre en el 100 % de los casos, cefalea en el 80 %, mialgias y artralgias en el 65 %, edema facial en el 55 %, y dolor abdominal en el 37,5 %. El tiempo promedio de incubación fue de 17 (3-21) días. El índice de infestación de Rhodnius prolixus fue de 3,3 % en el domicilio y de 2,2 % en el peridomicilio. En los cinco restaurantes inspeccionados, se encontraron condiciones sanitarias deficientes y alimentos con niveles de contaminación microbiológica inaceptables. Por último, un perro y dos zarigüeyas fueron positivos para los anticuerpos IgG en la prueba ELISA. Conclusiones. Mediante el análisis de las características epidemiológicas, ambientales y sanitarias, se confirmó un brote agudo de enfermedad de Chagas por exposición ocupacional y de posible transmisión oral, que podría ser el de mayor proporción reportado hasta la fecha en Colombia.

ABSTRACT Introduction: Trypanosoma cruzi, the etiological agent for Chagas disease, can be transmitted by oral intake of contaminated food or drinks. During epidemiological week 14 of 2014, two cases of acute Chagas disease were notified among hydrocarbons sector workers in Paz de Ariporo, Casanare. Objective: To characterize the affected population, to establish control and prevention measures and to confirm the outbreak. Materials and methods: We conducted an outbreak investigation that included the following components: a) Search for symptomatic people compatible with Chagas disease according to the case definition for their referral to medical services; b) entomological survey (192/197 houses); c) sanitary inspection and microbiological analysis of food samples; and d) study of reservoirs. Data management and analysis were done with Epi-Info 7.1.5 using descriptive statistics. We also calculated intradomicile and peridomicile triatomine infestation indexes. Results: We detected 552 exposed people; 40 had the disease (7.2%), of whom seven were women (17,5%) and 33, men (82.5%), i.e., a male-female ratio of 5:1. The mean age was 39.1 ± 10.8 years; the attack rate was 7.2% and lethality, 5% (2/40). Symptoms included fever (100% of cases), headache (80%), myalgia and arthralgia (65%), facial edema (55%), and abdominal pain (37.5%). The mean incubation time was 17 days (range: 3-21). Rhodnius prolixus domiciliary infestation index was 3.3 % and 2.2% in the peridomicile. In the five restaurants inspected sanitary conditions were deficient and food samples were microbiologically non-conforming. We found a dog and two opossums positive for IgG antibodies by ELISA. Conclusions: Environmental, sanitary and epidemiological conditions at the place confirmed an outbreak of Chagas diseases related to occupational exposure, possibly by oral transmission, which may be the largest to date in Colombia.

[Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia].

INTRODUCTION: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study.Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. MATERIALS AND METHODS: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. RESULTS: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor;however, the dwellings with report of triatomines were located nearer to the adjacent forest. CONCLUSIONS: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

[Possible oral transmission of Chagas disease among hydrocarbons sector workers in Casanare, Colombia, 2014].

INTRODUCTION: Trypanosoma cruzi, the etiological agent for Chagas disease, can be transmitted by oral intake of contaminated food or drinks. During epidemiological week 14 of 2014, two cases of acute Chagas disease were notified among hydrocarbons sector workers in Paz de Ariporo, Casanare. OBJECTIVE: To characterize the affected population, to establish control and prevention measures and to confirm the outbreak. MATERIALS AND METHODS: We conducted an outbreak investigation that included the following components: a) Search for symptomatic people compatible with Chagas disease according to the case definition for their referral to medical services; b) entomological survey (192/197 houses); c) sanitary inspection and microbiological analysis of food samples; and d) study of reservoirs. Data management and analysis were done with Epi-Info 7.1.5 using descriptive statistics. We also calculated intradomicile and peridomicile triatomine infestation indexes. RESULTS: We detected 552 exposed people; 40 had the disease (7.2%), of whom seven were women (17,5%) and 33, men (82.5%), i.e., a male-female ratio of 5:1. The mean age was 39.1 ± 10.8 years; the attack rate was 7.2% and lethality, 5% (2/40). Symptoms included fever (100% of cases), headache (80%), myalgia and arthralgia (65%), facial edema (55%), and abdominal pain (37.5%). The mean incubation time was 17 days (range: 3-21). Rhodnius prolixus domiciliary infestation index was 3.3 % and 2.2% in the peridomicile. In the five restaurants inspected sanitary conditions were deficient and food samples were microbiologically non-conforming. We found a dog and two opossums positive for IgG antibodies by ELISA. CONCLUSIONS: Environmental, sanitary and epidemiological conditions at the place confirmed an outbreak of Chagas diseases related to occupational exposure, possibly by oral transmission, which may be the largest to date in Colombia.

Enfermedad de Chagas de transmisión oral / Orally-transmitted Chagas disease

La enfermedad de Chagas es una zoonosis causada por el parásito protozoario Trypanosoma cruzi, transmitido con mayor frecuencia por vía vectorial. En los últimos años, sin embargo, se está observando un aumento marcado de la transmisión de la enfermedad por vía oral, asociada al consumo de bebidas preparadas a base de frutas u otros vegetales contaminados con las heces de triatominos o secreciones de mamíferos infectados. Después de un período de latencia de 3-22 días, a partir de la ingestión, la infección oral se caracteriza por manifestaciones más graves que la vectorial: fiebre prolongada, miocarditis aguda con insuficiencia cardíaca y en algunos casos meningoencefalitis. La mortalidad puede llegar hasta un 33% de los infectados. El objetivo de este trabajo es realizar una revisión del fenómeno y promover prácticas de prevención (AU)

Chagas disease is a zoonosis caused by protozoan parasite Trypanosoma cruzi, which is most frequently associated with a vectorial transmission. However, in recent years we have observed a significant increase in the oral transmission of the disease, associated mainly with the consumption of drinks made from fruit or other vegetables contaminated with triatomine faeces or secretions from infected mammals. After a latency period of 3 to 22 days after ingestion, the oral infection is characterized by more severe manifestations than those associated with vectorial transmission: prolonged fever, acute myocarditis with heart failure and, in some cases, meningoencephalitis. Mortality can reach up to 33% of those infected. The aim of this paper is to review this matter and to promote prevention practices (AU)

Riesgo de transmisión de la enfermedad de Chagas por intrusión de triatominos y mamíferos silvestres en Bucaramanga, Santander, Colombia / Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia

Introducción: La notificación de triatominos en las viviendas de algunos barrios de Bucaramanga motivó la realización de este estudio. Objetivo: Evaluar la intrusión de triatominos y mamíferos, así como algunos factores de riesgo para la enfermedad de Chagas en viviendas urbanas. Materiales y métodos: En un barrio de Bucaramanga, Santander, se recolectaron triatominos mensualmente durante un año con participación comunitaria mediante búsqueda manual en el alumbrado público, y el uso de trampas de luz, cebo animal y atrayentes químicos en el bosque cercano. Los reservorios se recolectaron con trampas cebadas. Los insectos y mamíferos se determinaron y examinaron para establecer su infección natural. Los factores de riesgo de las viviendas se midieron mediante una encuesta sobre factores sociales y ambientales. Resultados: Se recolectaron 11 adultos de Panstrongylus geniculatus y 63 de Rhodnius pallescens en el bosque, en zonas de recreación en el peridomicilio y en el domicilio, incluidas dos hembras y 21 ninfas de R. pallescens en dormitorios. Se capturaron dos ejemplares de Didelphis marsupialis en el bosques adyacente. De los 11 individuos de P. geniculatus capturados, se examinaron nueve, de los cuales cinco fueron positivos para Trypanosoma cruzi (56 %); de los 63 individuos de R. pallescens capturados, se examinaron ocho, cuatro de los cuales fueron positivos para T. cruzi (50 %). De dos especímenes de D. marsupiales capturados, uno fue examinado y se encontró que era positivo para T. cruzi. No se pudo establecer un factor de riesgo significativo, sin embargo, las viviendas con reporte de triatominos se encontraban más cerca del bosque adyacente. Conclusiones: El hallazgo de especies de triatominos intrusivas y de mamíferos con T. cruzi en el domicilio y el peridomicilio, así como en los bosques periurbanos, demuestra el riesgo de infección en las poblaciones que habitan en viviendas urbanas adyacentes a los ecótopos donde se mantiene el ciclo silvestre.

Abstract Introduction: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study. Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. Materials and methods: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. Results: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor; however, the dwellings with report of triatomines were located nearer to the adjacent forest. Conclusions: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

Situación de la infección por Trypanosoma cruzi en el oriente de Venezuela / Trypanosoma cruzi infection in Eastern Venezuela

Se advierte que, en la región oriental de Venezuela, deben ampliarse los estudios seroepidemiológicos para la enfermedad de Chagas ya que existen todas las variables de riesgo para el establecimiento de la transmisión vectorial de T. cruzi. (AU)

Situación de la infección por Trypanosoma cruzi en el oriente de Venezuela / Trypanosoma cruzi infection in Eastern Venezuela

Se advierte que, en la región oriental de Venezuela, deben ampliarse los estudios seroepidemiológicos para la enfermedad de Chagas ya que existen todas las variables de riesgo para el establecimiento de la transmisión vectorial de T. cruzi.

Subdominant/cryptic CD8 T cell epitopes contribute to resistance against experimental infection with a human protozoan parasite.

During adaptive immune response, pathogen-specific CD8(+) T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8(+) T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8(+) T cells of H-2(a) infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8(+) T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8(+) T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.

Análisis cuantitativo del crecimiento y cambio morfométrico en poblaciones de Leishmania chagasi y Trypanosoma cruzi mantenidos en cultivos axénicos puros y mixtos / Quantitative approach to the analysis of the growth and morphometric change in populations of Leishmania chagasi and Trypanosoma cruzi maintained in pure and mixed axenic cultures

Las relaciones que se establecen entre géneros de la familia trypanosomatidae en condiciones de coexistencia en el mismo medioambiente pueden estar vinculadas a respuestas compensatorias inter-poblacionales que incluyen cambios morfológicos (diferentes estadios) y morfométricos (diferencias mensurables). El análisis cuantitativo de tales respuestas en cultivos axénicos puros de Leishmania chagasi y trypanosoma cruzi, así como en isomezclas axénicas de L. chagasi-T. cruzi mantenidas in vitro, no ha sido abordado, desconociéndose por lo tanto, particularidades biológicas. Muestras interdiarias de cultivo se fijaron, colorearon, observaron, digitalizaron y procesaron cuantitativamente. Además de cuantificar las densidades poblacionales, se registraron las magnitudes numéricas de variables morfométricas que, posteriormente, se analizaron con herramientas estadísticas. Los resultados indicaron cambios específicos en las variables investigadas, así como heterogeneidad morfométrica entre los mismos morfotipos de los mismos géneros al ser mantenidos en cultivos puros o mixtos. Los modelos de cambio morfométrico de L. chagasi y T. cruzi en cultivos puros difieren de los modelos de cambio morfométrico en los cultivos mixtos (L. chagasi-T. cruzi). Las metodologías biométricas discriminan, en términos morfométricos, poblaciones del mismo estadio (morfotipo) en ambientes diferentes.

The relations established among genera of the Trypanosomatidae family in coexisting conditions in the same environment may be linked to inter-population compensatory answers that include morphological (differences among stages) and morphometrical (measurable difference) changes. The quantitative analysis of these answers in Leishmania chagasi and Trypanosoma cruzi pure axenic cultures, as well as in L. chagasi - T. cruzi axenic iso-mixtures in vitro maintained has not been approached, and consequently, potentially useful biological particularities in the control of these important human parasites are unknown. Every other day culture samples were fixed, stained, observed, digitalized and quantitatively processed. In addition to quantify, the population densities and the appearance-disappearance stage (morphotypes) dynamics, the numeric magnitudes of the morphometric variables were recorded and later analyzed with multivariate statistical techniques. The results indicate specific changes in the investigated variables, as well as morphometric heterogeneity between the same morphotypes of the same genera when maintained in pure or mixed cultivation. The morphometric change models for L. chagasi and T. cruzi in pure culture differ from the models of morphometric change in mixed cultivation (L. chagasi-T. cruzi). The biometric methodologies discriminate in morphometric terms populations of the same stage (morfotype) in different environments.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Some snake venom proteins present enzymatic activities, such as L-amino acid oxidase (LAAO). The aim of this paper was to investigate the effect of Bothrops leucurus total venom (BleuTV) and its fraction LAAO (BleuLAAO) on bacteria, yeast, and promastigote forms of Leishmania amazonensis and Leishmania chagasi, and epimastigote forms of Trypanosoma cruzi. BleuTV was isolated with a Protein Pack 5PW® (Waters Corporation, USA), and several fractions were obtained. BleuLAAO was purified to high molecular homogeneity, and its N-terminal amino acid sequence shared a high degree of amino acid conservation with other LAAOs. BleuTV inhibited Staphylococcus aureus growth in a dose-dependent manner, with a minimum inhibitory concentration (MIC) of 25 ìg/mL, which corresponded to its minimum lethal concentration (MLC). BleuTV also inhibited the growth of promastigote forms of L. chagasi and L. amazonensis, with respective IC50 values of 1.94 ìg/mL and 5.49 ìg/mL. Furthermore, it repressed T. cruzi growth with an IC50 of 1.14 ìg/mL. However, BleuLAAO did not inhibit the growth of the microorganisms studied and was not toxic to macrophages. BleuTV had low toxicity against macrophages at the concentrations studied. In conclusion, whole venom from Bothrops leucurus inhibited the growth of some microorganisms, including S. aureus, Leishmania sp., and T. cruzi.

Anomalías morfológicas y estructurales en fetos de ratones con infección aguda experimental por Trypanosoma cruzi / Morphological and structural anomalies in fetuses of mice with acute experimental infection by Trypanosoma cruzi

El presente estudio muestra el desarrollo de fetos de ratones hembras NMRI inoculadas con la cepa M/HOM/BRA/53/Y de trypanosoma cruzi y preñadas durante la fase aguda de la infección. Altos niveles de parasitemias fueron observados en las ratones con 30 días post-infección y 20 días de gestación, en comparación con las detectadas en los ratones vírgenes e infectadas con T. cruzi. En 3 de los fetos (15%) provenientes de dos madres infectadas/gestantes con altas parasitemias, se observaron signos de anomalías congénitas morfológicas y estructurales músculo-esqueléticas. Presentándose en uno de los fetos la formación de dos protuberancias, una sobre el lado dorsal del cuerpo y la otra en la base de la pata inferior izquierda, en otro de los fetos, la pata derecha se desarrolló sobre el lado derecho de la cara y en un tercer feto se formó una protuberancia en la pata anterior izquierda a nivel de la región subescapular. El estudio histopatológico con hematoxilina y eosina de los tejidos muscular esquelético y cardíaco, mostró en el 10% (2/20) de los tejidos fetales, intenso infiltrado celular mononuclear con predominio de linfocitos, macrófagos o histiocitos y monocitos entre las fibras musculares y cardíacas, con discreta miositis y miocarditis. Con la técnica de peroxidada anti peroxidada se observaron abundantes depósitos antigénicos, tanto en placenta como en músculo esquelético de los fetos con alteraciones morfológicas. En los ratones infectadas/gestantes los fetos presentaron reducción del peso corporal y retardo en el crecimiento fetal, así como reducción en el número de fetos de 10 en comparación con 14 fetos de mayor tamaño y aspecto normal desarrollados en las ratonas sanas preñadas.

The present study shows the development of the fetuses from pregnant female mice NMRI inoculated with M/HOM/BRA/53/Y Trypanosoma cruzi strain. The infection revealed the highest levels of patent parasitemia in mice with 30 days postinfection and 20 days of pregnancy in comparison with infected unmated mice. Three fetuses (15%) from two infected mice with high levels of parasitemia, showed morphological and structural muscularskeletal congenital anomalies. Two protuberances were observed, one on the dorsal side of the body, and the other on the left footpad base. In another fetus his right footpad came out from the right part of his face and in the 3rd one it was observed a lump in the left leg, above the level subscapular region. The histophatological study with hematoxilin-eosin staining of skeletal muscle and cardiac tissue, showed in 10% (2/20) of the mice, inflammatory infiltrate with lymphocytes, macrophages and monocytes into muscular and cardiac fibers, with discrete myositis and myocarditis. Peroxidase anti-peroxidase staining showed T. cruzi antigens in placenta and skeletal muscle of the fetuses with morphological alterations. In the pregnant mice, fetuses also showed both, loss of weight and growth retardation, as well as reduction of the number of fetuses to 10 in comparison with 14 fetuses in normal and healthy pregnant mice.

Susceptibilidad de trypanosoma cruzi a diferentes venenos de serpientes de Costa Rica / Susceptibility of Trypanosoma cruzi to different Costa Rican snake venoms

Se evaluaron los efectos tóxicos de los venenos de cinco serpientes costarricenses en cuanto a su capacidad tripanocida contra dos cepas de trypanosoma cruzi y sus efectos en cuanto a los mecanismos de muerte celular. Los venenos de bothrops asper, bothriechis schlegelii, crotalus durissus durissus, atropoides nummifer y A. picadoi, mostraron actividad tripanocida contra las formas de epimastigoto, amastigoto y tripomastigoto. Los venenos de b. asper y de A. nummifer presentaron la más alta citotoxicidad para las células Vero. Los de b. asper y b. schlegelii presentaron la más alta actividad en los epimastigotos de la cepa CL, mientras que los venenos de b. asper y el de A. nummifer fueron más eficientes contra los epimastigotos de la cepa Jennifer. El veneno de b. schlegelii produce un efecto proliferativo en las células Vero; mientras que el de C. d. durissus produce el mismo efecto en los epimastigotos de la cepa CL, ambos a la concentración de 2,5 Ig/mL. Los valores de CI50 mostraron que se requieren menores cantidades contra los amastigotos en relación con los epimastigotos. Los venenos de b. asper y B. schlegelii presentan la más alta actividad contra los amastigotos de ambas cepas. Con los tripomastigotos sanguíneos de la cepa GA, los cinco venenos ocasionaron una disminución de la motilidad en los diferentes tiempos de exposición, pero el veneno de A. nummifer, en las concentraciones más bajas, mostró una actividad más marcada en comparación con los otros veneno. En cuanto a los efectos de los venenos, mediados por los grados de apoptosis, necrosis o proliferación celular, se observó que estos fenémenos se presentan y tienen relación con el tipo de veneno, su concentración y el tiempo de exposición.

The trypanocide effect of venoms from five Costa Rican species of snakes was evaluated against two strains of trypanosoma cruzi and their cellular toxic effects were likewise observed. The venoms of Bothrops asper, bothriechis schlegelii, crotalus durissus durissus, atropoide nummifer and A. picadoi showed evident trypanocide action against epimastigotes, amastigotes and trypomastigotes. The venoms of b. asper and b. schlegelii were shown to be the most active against the epimastigotes of the CL strain, whereas those of b. asper and A. nummifer were more effective against the epimastigotes of the Jennifer strain. The venoms of b. schlegelii and C.d. durissus, at the lowest concentrations of 2.5 Ig/mL, were able to trigger a proliferative effect on Vero cells and epimastigotes of the CL strain, respectively. The IC50 values showed that lower amounts of venoms are necessary in order to inhibit amastigotes as compared to epimastigotes. The venoms of b. asper and b. schlegelii exhibited the highest activity against amastigotes of both T. cruzi strains. All venoms were able to arrest motility of blood trypomastigotes of the GA strain at different times and the most active in this case was A. nummifer venom. The toxic effects of the venoms measured by the degree of apoptosis, necrosis and cell proliferation that they produced showed that all these events occur and are related to the type of venom, its concentration and exposure time.

Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes.

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.

Análisis de la composición poblacional de aislados venezolanos de Trypanosoma cruzi I, provenientes de un hospedador humano, un insecto triatomineo y un reservorio natural / Analysis of the population composition of venezuelans isolates of Trypanosoma cruzi I fron a human host, a triatomine insect and a nature reservoir

Se analizó la composición poblacional de aislados venezolanos pertenecientes al grupo Tel, provenientes de distintos hospedadores. El análisis fue realizado mediante la clonación de los aislados utilizando la técnica de plaqueo, de la cual se originaron clones de cada uno que posteriormente fueron comparados mediante la técnica de Amplificación Aleatoria de ADN Polimórfico (RAPD). La observación de los perfiles de bandas obtenidos mediante electroforesis en gel de agarosa, mostró diferencias en cuanto al número y la posición de bandas en los clones obtenidos de los aislados provenientes de humano y del reservorio natural (Dildelphis marsupiales), mientras que para el aislado proveniente del vector (Triatoma ningromaculata), todos los clones generaron perfiles de bandas similares. Estos resultados evidencian una heterogeneidad poblacional para el caso de los dos primeros aislados. El índice de variabilidad genética fue diferente para cada uno de los aislados, siendo mayor para dos aislados provenientes de dos pacientes chagásicos, mostrando un valor de 0.1652 y 0.0853 respectivamente, mientras que para el aislado proveniente del reservorio natural, se obtuvo un valor de 0.0312. Estos resultados indican polimorfismo existente entre clones provenientes de un mismo aislado, siendo indicativo de la existencia de heterogeneidad intrapoblacional, lo cual pudiese tener implicaciones en la diversidad de manifestaciones clínicas reportadas para infecciones con T. cruzi I.

It was analyzed the population composition of Venezuelan isolates belonging to the genotype Tel from different hosts. The analysis was carried out through the cloning of the isolates using the plating technique, obtaining clones of each one of them, which were afterward compared using the random amplified plymorphic DNA technique (RAPD). The observation of bands'profiles obtained by agarose gel electrophoresis, showed differences regarding to the number and position of the bands between the isolates from both humans and natural reservoir (Dildelphis marsupialis), except for the clones obtained from the vector isolate (triatoma nigromaculata), where all of them gave the same pattern of bands. This result reveals the presence of population heterogeneity for the isolates from human and natural host. The genetic variability index was different in each one of the isolates, being higher for the isolates fron two chagasic patientes, showing values of 0.1652 and 0.0853 respectivivly; and for the isolate fron the natural reservoir the index value was 0.0312. These results reveals the presence of polymorphism between the clones obtained from the same isolate, showing the existence of intra popultaion heterogenicity, wich might have implications on the diversity of clinical manifestations previously reported with T. cruzi I infections.

Trypanosoma cruzi: ultrastructural studies of adhesion, lysis and biofilm formation by Serratia marcescens.

A few days after blood meal the number of bacteria in the anterior midgut (stomach) of Rhodnius prolixus, a vector of Trypanosoma cruzi, the causative agent of Chagas' disease, increases dramatically. Many of the bloodstream trypomastigotes of the pathogenic protozoan as well as ingested erythrocytes are lysed in the stomach. Incubation of T. cruzi with Serratia marcescens variant SM365, lead to parasite lysis. In the present study, this bacterium rapidly adhered to the protozoan surface through d-mannose recognizing fimbriae and rapidly induced its complete lysis. In contrast, the DB11 variant of the same bacterial species did not adhere and did not induce protozoan lysis. Scanning and transmission electron microscopy revealed that following bacteria-protozoan attachment there is an assembly of long filamentous structures, identified as a biofilm, which connect the protozoan to the bacteria forming bacterial clusters. We conclude that parasite lysis and biofilm formation mechanisms are important for understanding parasite-microbiota interactions in the gut of insect vectors of trypanosomatids.

Inhibitory effects of d-mannose on trypanosomatid lysis induced by Serratia marcescens.

Studies were carried out on the effects of different carbohydrates on the lysis of Trypanosoma cruzi, Trypanosoma rangeli and erythocytes caused by the bacteria Serratia marcescens variants SM 365 and RPH. High concentrations of d-mannose were found to protect T. cruzi and T. rangeli markedly diminishing the lysis caused by S. marcescens. However, this carbohydrate is unable to interfere with the hemolysis induced by SM 365 and RPH variants. These results showed that the trypanolytic effect induced by S. marcescens SM 365 and RPH variants is dependent on d-mannose and distinct from the hemolytic activity, strongly suggesting that bacterial fimbriae are relevant to S. marcescens in lysis of parasites.