ABSTRACT
Introducción. La creciente resistencia bacteriana a los antibióticos representa una amenaza mundial de salud pública. Las excreciones y secreciones larvarias derivadas de moscas necrófagas de la familia Calliphoridae podrían configurar una fuente promisoria para contrarrestar sus efectos. Objetivo. Comparar la actividad antimicrobiana de las excreciones y secreciones larvarias nativas, y de las mayores y menores de 10 kDa de Calliphora vicina y Sarconesiopsis magellanica (Diptera: Calliphoridae). Materiales y métodos. El bioensayo se hizo a partir de la técnica de turbidimetría y en el caso de las excreciones y secreciones menores de 10 kDa se determinó la concentración inhibitoria mínima (CIM). Resultados. Las excreciones y secreciones nativas y las menores de 10 kDa de C. vicina y S. magellanica, evidenciaron una potente actividad antibacteriana contra tres cepas de Staphylococcus aureus y cuatro bacterias Gram negativas, siendo las menores de 10 kDa más efectivas que las nativas en las dos especies de moscas evaluadas. Además, las menores de 10 kDa presentaron la misma efectividad, aunque en las pruebas de CIM se observó que las de S. magellanica fueron más potentes en todas las bacterias evaluadas, excepto contra la cepa de S. aureus ATCC 25923. Las mayores de 10 kDa no inhibieron el crecimiento bacteriano. Conclusión. Los resultados validaron, en general, que estas sustancias son fuente importante para el aislamiento y la caracterización de agentes antimicrobianos.
Introduction: The growing resistance to antibiotics worldwide represents a global threat to public health. The larval excretions and secretions derived from necrophagous flies from the Calliphoridae family could represent a promising source for counteracting their effects. Objective: To compare the antimicrobial activity of Calliphora vicina and Sarconesiopsis magellanica (Diptera: Calliphoridae) native excretions and secretions and those weighing more than 10 kDa and less. Materials and methods: We used the turbidimetry technique for the bioassay; we determined the minimum inhibitory concentration (MIC) for excretions and secretions weighing less than 10 kDa. Results: Calliphora vicina and S. magellanica native excretions and secretions and those weighing less than 10 kDa exhibited potent antibacterial activity against three Staphylococcus aureus strains and four Gram-negative bacteria; those weighing less than 10 kDa were more effective than the native ones in the two species of flies evaluated here. Furthermore, excretions and secretions weighing less than 10 kDa had the same effectiveness, except in the MIC trials where S. magellanica excretions and secretions weighing less than 10 kDa were more potent against all the bacteria evaluated, except for S. aureus ATCC 25923. Excretions and secretions weighing more than 10 kDa did not inhibit bacterial growth. Conclusions: These results potentially validate these substances as an important source for isolating and characterizing antimicrobial agents.
Subject(s)
Modalities, Secretion and Excretion , Diptera , Gram-Negative Bacteria , Gram-Positive Bacteria , Larva , Anti-Bacterial AgentsABSTRACT
BACKGROUND The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs' physico-chemical properties. Synthetic AMPs' antibacterial activity was evaluated. FINDINGS Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules' sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.
Subject(s)
Animals , Diptera , Fat Body , Microbial Sensitivity Tests , Pore Forming Cytotoxic Proteins , Calliphoridae , Larva , Anti-Bacterial Agents/pharmacologyABSTRACT
BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.
Subject(s)
Animals , Psychodidae/parasitology , Leishmania braziliensis/genetics , Proteomics , Leishmania/genetics , Cell Line , TranscriptomeABSTRACT
Objetivo: Evaluar, en condiciones in vitro , la actividad antibacterial de los extractos de cuerpos grasos y de la hemolinfa de larvas de tercer estadio de Sarconesiopsis magellanica , la cual se comparó con los efectos obtenidos de las mismas sustancias derivadas de Lucilia sericata . S. magellanica (Diptera: Calliphoridae) es una mosca de importancia principalmente forense, utilizada en la determinación del intervalo post mortem . Por sus hábitos necrófagos, es considerada un modelo potencialmente útil en terapia larval. Material y métodos: Se extrajeron los cuerpos grasos de las larvas mediante la técnica de disección corporal y la hemolinfa se obtuvo mediante decapitación y centrifugación de los especímenes larvales. Las bacterias evaluadas fueron Staphylococcus aureus y Pseudomonas aeruginosa . Los métodos utilizados para evaluar la actividad antibacterial fueron difusión en agar y unidades formadoras de colonias (UFC/ml). Resultados: Después de la correspondiente incubación, los resultados generales mostraron que la actividad antibacterial de la hemolinfa y de los cuerpos grasos, tanto de L. sericata como de S. magellanica , fueron efectivos contra S. aureus y P. aeruginosa sin diferencias significativas entre las especies de moscas, aunque con algunas diferencias entre las cepas bacterianas. Conclusiones: Los resultados obtenidos sugieren que estas sustancias podrían tener un efecto similar en el tratamiento de heridas infectadas contra los microorganismos evaluados.
Objective: This study aimed to evaluate the in vitro antibacterial activity of fat body and hemolymph extracts from Sarconesiopsis magellanica (Diptera: Calliphoridae) third-instar larvae, compared to the effect obtained using the same extracts but derived from Lucilia sericata . S. magellanica blowflies are considered important in forensic sciences due to their usefulness in determining the post mortem interval. This blowfly could be useful in larval therapy due to its necrophagous habits. Materials and methods: Fat body from larvae was removed by dissection, and hemolymph via decapitation and centrifugation of larval specimens. The antibacterial effect was tested against Staphylococcus aureus and Pseudomonas aeruginosa using two methods: agar diffusion and colony forming units (CFU/mL). Results: Hemolymph and fat body extracts derived from both L. sericata and S. magellanica were effective against S. aureus and P. aeruginosa , with no significant differences between blowfly species, although with some differences between the bacterial strains. Conclusions: The results obtained suggest that S. magellanica and L. sericata fat body and hemolymph extracts might have a similar antimicrobial activity against these microorganisms when used to treat infected wounds.
Subject(s)
Humans , Animals , Staphylococcus aureus , Pseudomonas aeruginosa , Bacteria , In Vitro Techniques , Cross Infection , Diptera , Infections , LarvaABSTRACT
Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.
Subject(s)
Animals , Cell Culture Techniques/methods , Culex/cytology , Embryo, Nonmammalian/cytology , Cell Proliferation , Cell Adhesion/physiology , Cell Line/chemistry , Cell Line/cytology , Karyotype , Polymerase Chain ReactionABSTRACT
The objective of this work was to establish, under experimental laboratory conditions, a colony of Lucilia sericata, Bogotá-Colombia strain, to build life tables and evaluate two artifcial diets. This blowfy is frequently used in postmortem interval studies and in injury treatment. The parental adult insects collected in Bogotá were maintained in cages at 22°C±1 average temperature, 60 percent±5 relative humidity and 12 h photoperiodicity. The blowfies were fed on two artifcial diets that were evaluated over seven continuous generations. Reproductive and population parameters were assessed. The life cycle of the species was expressed in the number of days of the different stages: egg = 0.8±0.1, larvae I = 1.1±0.02, larvae II = 1.94±0.16, larvae III = 3.5±0.54, pupae = 6.55±0.47, male adult = 28.7±0.83 and female adult = 33.5±1.0. Total survival from egg stage to adult stage was 91.2 percent for diet 1, while for diet 2 this parameter was 40.5 percent. The lifetime reproductive output was 184.51±11.2 eggs per female. The population parameters, as well as the reproductive output of the blowfies that were assessed, showed relatively high values, giving evidence of the continuous increase of the strain over the different generations and making possible its maintenance as a stable colony that has lasted for more than two years.
Subject(s)
Animals , Female , Male , Diet , Diptera/physiology , Life Tables , Life Cycle Stages/physiology , Animals, Laboratory , Colombia , Diptera/classificationABSTRACT
El propósito principal de la investigación aquí presentada fue obtener cultivos celulares primarios derivados de Lucilia sericata (Diptera: Calliphoridae). Esta mosca necrófaga es utilizada para determinar el intervalo post-mortem y en terapia larval. A partir de huevos embrionados, se realizaron explantes en diversos medios de cultivo (Grace, Schneider, MM/VP12, DMEM, Grace/L-15 y L-15), suplementados con 20% de suero fetal bovino. La esterilización del material biológico se efectuó mediante la aplicación de soluciones de formaldehido e hipoclorito de sodio. El crecimiento celular se inicio en los medios L-15, MM/VP12, Grace/L-15 y Schneider, en un tiempo promedio de 10 días después de efectuadas las siembras de tejidos embrionarios, mediante la proliferación de grupos de colonias dispersas en la superficies de los frascos de cultivo y a partir de las terminaciones de los fragmentos larvales. La evolución del crecimiento celular hasta la formación de la monocapa semiconfluente fue relativamente rápida, se alcanzo a las tres semanas post-explantes. La morfología de las células en los cultivos fue heterogénea, se destacaron formas epitelioides, similares a nerviosas, gigantes e irregulares. La comparación de las características de crecimiento de los cultivos celulares de L. sericata con los obtenidos de otras especies de dípteros mostro mayor favorabilidad en la evolución, en razón a que las células se adaptaron mejor a las condiciones fisico-quimicas de varios medios de cultivo. Este es el primer informe de cultivos celulares de una mosca de la familia Calliphoridae.
The main purpose of this study was to obtain primary cell cultures derived from Lucilia sericata (Diptera: Calliphoridae). Necrophagous this fly is used for determination of post-mortem interval and larval therapy. Since explants embryonated eggs were performed in various culture media (Grace Schneider, MM/VP12, DMEM, Grace/L-15 and L-15), supplemented with 20% fetal serum. Sterilization of the biological material was carried out by immersing it in formaldehyde and sodium hypochlorite solutions. The cell growth was initiated in the L-15, MM/VP12, and Schneider Grace/L-15 in an average time of 10 days after completion of planting by the proliferation of groups of colonies scattered on the surface of the boxes crops and also from the endings of larval fragments. The evolution of cell growth to the formation of monolayer semi-confluent was relatively fast, reaching at 3 weeks post-explant. Cellular morphology in cultured cells was heterogeneous, especially epithelioid forms, similar to nerve, giant and irregular. Comparison of the growth characteristics of these cell cultures with those obtained from other species of flies was more favorable in the evolution of those obtained from L. sericata, on the grounds that the cells are better adapted to the physical-chemical conditions of several culture media. This is the first report of a cell culture-fly family Calliphoridae.
Subject(s)
Humans , Calliphoridae , Cells, Cultured , Diptera , Research Report , Primary Cell CultureABSTRACT
En el presente trabajo se evaluó la actividad tóxica de extractos de Eupatorium microphyllum L.F. sobre larvas de IV estadio del mosquito Aedes aegypti (Linneaus), bajo condiciones de laboratorio. Se utilizaron extractos acuosos en concentraciones del 500 mg L-1, 1.500 mg L - 1 y 2.500 mg L-1 y acetónicos en concentraciones de 10 mg L-1, 20 mg L-1, 30 mg L-1, 40 mg L-1 y 50 mg L-1. Los bioensayos se realizaron por triplicado, cada uno con 20 larvas, expuestas durante 24 horas a 150 mL de solución. En todos los ensayos biológicos se emplearon grupos control. En la evaluación de los extractos acetónicos, se empleó un control negativo para evitar que la mortalidad de las larvas ocurriera a causa del solvente. Los extractos acuosos mostraron acción moderadamente baja en la mortalidad de larvas, menor del 20%. Por el contrario, la acción de los extractos acetónicos se observó a 10 y 20 mg L-1, con 15% de mortalidad, mientras que a 30 y 40 mg L-1 se registraron 22 al 38% de mortalidad, en tanto que a 50 mg L-1 la mortalidad fue del 95,4% con resultados estadísticos altamente significativos. Las concentraciones de los extractos acetónicos mostraron ser las más eficientes para el control de los mosquitos seleccionados. Ambos tipos de extractos mostraron efecto tóxico en larvas de A. aegypti ; sin embargo, se observó mayor efecto en los extractos acetónicos en relación con los extractos acuosos de E. microphyllum, lo cual constituye una alternativa viable en la búsqueda de nuevos larvicidas a partir de compuestos naturales.
In the present work the toxic activity of extracts of Eupatorium microphyllum L.F. was evaluated on 4 th instar larvae of the mosquito Aedes aegypti (Linneaus), under laboratory conditions. Aqueous extracts were utilized in concentrations of 500 mg L-1, 1,500 mg L-1 and 2,500 mg L-1 and acetone in concentrations of 10 mg L-1, 20 mg L-1, 30 mg L-1, 40 mg L-1 and 50 mg L-1. The bioassays were carried out for triplicate each one with 20 larvae, exposed for 24 hours to 150 mL of solution. In all the bioassays were employed control groups. In the evaluation of the acetone extracts, a negative control was employed to avoid that the mortality of the larvae to occur on account of the solvent. The Aqueous extracts showed low moderate action in the mortality of larvae, less than 20%. On the contrary, the action of the acetone extracts was observed to 10 and 20 mg L-1 with 15% of mortality, while to 30 and 40 mg L-1 were registered 22 to 38% of mortality. However, to 50 mg L-1 the mortality was of 95.4% with highly significant statistical results. The concentrations of the acetone extracts showed to be the most efficient for the control of the mosquitoes selected. Both types of extracts showed toxic effect in larvae of A. aegypti, nevertheless, greater effect in the acetone extracts was observed relating to the aqueous extracts of E. microphyllum, which constitutes a viable alternative in the search of new larvicides from composed natural.
Subject(s)
Animals , Eupatorium , Solutions , Solvents , Biological Assay , Aedes , ToxicityABSTRACT
Introducción. Durante las últimas dos décadas, la terapia larval ha resurgido como una alternativa confiable y segura para la cura de úlceras cutáneas que no responden a los tratamientos convencionales. Objetivo. Evaluar el uso de las larvas de Lucilia sericata en el tratamiento de heridas infectadas con Pseudomonas aeruginosa en un modelo animal. Materiales y métodos. Se tomaron 12 conejos, los cuales fueron divididos al azar en 3 grupos homogéneos: al primero se le aplicó terapia larval, el segundo se trató con terapia antibiótica y el tercero fue establecido como control. A cada uno de los animales se les realizó una herida, luego se inoculó en ésta una suspensión de P. aeruginosa y, finalmente, al registrarse el desarrollo de la infección, se procedió, en los dos primeros grupos, a los tratamientos correspondientes. Para la evaluación macroscópica de las heridas, se tuvo en cuenta la presencia de edema y exudado, mal olor, inflamación alrededor de la herida y apariencia del tejido de granulación. Al proceso de cicatrización se le hizo seguimiento a través de una técnica dermohistológica. Resultados. Se registraron claras diferencias entre el grupo de animales tratados con terapia larval vs. el grupo tratado con terapia convencional de antibióticos, estableciéndose un periodo de 10 días para alcanzar la cicatrización en el grupo de terapia larval mientras que en el segundo grupo el proceso se cumplió en 20 días. Conclusiones. S e demostró la eficacia de las larvas de L. sericata en el tratamiento de heridas infectadas con P. aeruginosa.
Introduction. During the last two decades the larval therapy has reemerged as a safe and reliable alternative for the healing of cutaneous ulcers that do not respond to the conventional treatments. Objective. To evaluate the use of the larvae of Lucilia sericata as a treatment for infected wounds with Pseudomonas aeruginosa in an animal model. Materials and methods. Twelve rabbits were randomly distributed in 3 groups: the first group was treated with larval therapy; the second was treated with antibiotics therapy and to the third no treatment was applied, therefore was established as a control group. To each animal a wound was artificially induced, and then a suspension of P. aeruginosa was inoculated into the lesion. Finally, every rabbit was evaluated until the infection development was recognized and treatment was set up for the first two groups according with the protocols mentioned above. Macroscopic evaluation of the wounds was based on the presence of edema, exudates, bad odor, inflammation around the wound and the presence of granulation tissue. The healing process was evaluated by monitoring histological changes in the dermal tissue. Results. Differences in the time required for wound healing were observed between the first group treated with larval therapy (10 days) and the second group treated with conventional antibiotics therapy (20 days). Conclusion. The L. sericata larva is and efficient tool as a therapy for infected wounds with P. aeruginosa.
Subject(s)
Animals , Models, Animal , Pseudomonas aeruginosa , Therapeutics , Wound Healing , Wounds and Injuries , LarvaABSTRACT
Los cultivos celulares de mosquitos son frecuentemente utilizados para el aislamiento, identificación y caracterización de arbovirus. Para el estudio de los virus dengue (VDEN) y virus de fiebre amarilla (VFA) se emplean, principalmente, la línea celular C6/36 de Aedes albopictus y la línea celular AP61, obtenida de Aedes pseudoscutellaris. La línea celular de A. aegypti AEGY28, previamente obtenida a partir de tejidos embrionarios del vector, se utilizó en el presente trabajo para evaluar la susceptibilidad a la infección por VDEN y VFA. Para ello, los cultivos celulares se ensayaron a diferente multiplicidad de infección con los aislados clínicos de virus dengue tipo 2 (COL789, MOI: 1 y 5) y VFA (V341, MOI 0,02). Posteriormente se realizó la detección de antígenos virales por la técnica de inmunocitoquímica y su cuantificación por la técnica de CellELISA fluorométrica. Se usaron como controles positivos de infección, tanto células C6/36 como células VERO. Inesperadamente, no se observó inmunoreactividad en las células de A. aegypti infectadas con ambos tipos de virus en ninguno de los MOI o tiempos estudiados. Tampoco se evidenció antígeno por la técnica fluorométrica ni fue posible detectar RNA viral por RTPCR a partir de células infectadas. Por lo tanto, se puede concluir que la línea celular de A. aegypti no es susceptible a la infección por VDEN ni por VFA. Ello podría estar relacionado con características propias de la membrana celular o de la maquinaria enzimática necesaria para la replicación viral.
Mosquito cell derived cultures are useful tools for arbovirus isolation, identification or characterization. For studying dengue (DENV) and yellow fever viruses (YFV) Aedes albopictus C6/36 or Aedes pseudoscutellaris AP61 cell lines, are normally used. The Aedes aegypti AEGY28 cell line was obtained from embryonic tissues and characterized previously by one of us. In order to evaluate its susceptibility to two Flavivirus, AEGY28 cells were inoculated with different multiplicity of infection (MOI) with type 2 DENV (COL789, MOI: 1 and 5) and YFV clinical isolates (V341, MOI 0,02) then processed at different times post infection (p.i.). Immunostaining and fluorometric cellELISA were carried out to identify and quantify viral antigens. C6/36 and Vero cells were used as positive controls. Unexpectedly, immunoreactivity was not found in inoculated AEGY28 cells, even in higher MOI or late times p.i., therefore antigen quantification using fluorometric cellELISA were not plausible. Reverse transcriptase PCR with specific primers did not detect viral RNA in AEGY28 inoculated cells. We can conclude that Aedes aegypti AEGY28 cell line is not susceptible to dengue and yellow fever Flavivirus, a finding possibly related with the lacking of specific molecules at the plasma membrane or absence of cell machinery necessary for viral replication.
ABSTRACT
El flebótomo Lutzomyia spinicrassa es vector de Leishmania braziliensis y tiene amplia distribución en plantaciones de café en Colombia y Venezuela. Metodología: Se estableció una colonia en condiciones de laboratorio a partir de 600 hembras de L. spinicrassa capturadas en el campo y mantenidas a temperatura de 23º C y humedad relativa de 70%. El tiempo de desarrollo desde huevo hasta adulto osciló entre 58 y 78 días, en promedio 11 semanas. Se compararon parámetros poblacionales de la especie obtenidos a partir de cinco generaciones sucesivas mantenidas en grupos, con una generación criada individualmente. Resultados: Se obtuvieron los siguientes parámetros en cada condición experimental: tasa neta de reproducción (6,92 y 7 hembras por hembra por generación), tasa intrínseca de incremento poblacional (0,17 y 0,18 hembras por hembra por semana) y tasa finita de incremento poblacional (1,06 y 1,19 individuos por hembra por semana). Conclusión: Estos datos sugieren que la colonia de L. spinicrassa tuvo un incremento constante durante las seis generaciones analizadas.
Lutzomyia spinicrassa is a vector of Leishmania braziliensis. This sand fly has a broad geographical distribution in Colombia and Venezuela and it's founded mainly in coffee plantations. Methodology: Starting from 600 females of L. spinicrassa captured in field a laboratory colony was established. The development time from egg to adult ranged from 58 to 78 days, 11 weeks in average. Population parameters of five successive generations maintained in groups were compared with a generation reared individually. Results: The following parameters were obtained in each experimental condition: net rate of reproduction (6.92 and 7 females per female per generation), intrinsic rate of population increment (0.17 and 0.18 females per female per week) and finite rate of population increment (1.06 and 1.19 individuals per female per week). Conclusion: These data suggest that the colony of L. spinicrassa had a constant increment during the six analyzed generations.
Subject(s)
Animals , Psychodidae , Population , Leishmania braziliensis , DipteraABSTRACT
Se evaluó la susceptibilidad de los cultivos celulares derivados de tejidos embrionarios de Aedes aegypti a la infección con Leishmania (L) chagasi y Leishmania (V) braziliensis, agentes etiológicos de leishmaniasis visceral americana y leishmaniasis cutánea, respectivamente. Metodología: Se seleccionaron células de A. aegypti mantenidas en una mezcla de medio de cultivo Grace/L15, suplementado con suero fetal bovino al 15%, albendazol 5,4 mg/ml y una mezcla de antibióticos, e incubadas a una temperatura promedio de 26 °C. Los cultivos celulares fueron inoculados con promastigotes metacíclicos de la cepa MH/CO/84/CI-044B de L. chagasi y la cepa HOM/BR752903 de L. braziliensis en una concentración de 10 parásitos por célula. Como control positivo de la infección se utilizó la línea celular J774. Resultados: Los registros más altos en el porcentaje de infección y en el número de amastigotes por células en los cultivos celulares A. aegypti y en la línea celular J774 se obtuvieron en los días 6 y 9 pos-infección. Los resultados mostraron interacción, internalización y maduración in vitro de las dos especies del parásito en las células de este insecto no vector de Leishmania. Las células de A. aegypti infectadas mostraron cambios en el área por la influencia de los parásitos, contrario a lo registrado en las células no infectadas (P<0,05). Conclusión: Los cultivos celulares de A. aegypti emergen como un nuevo modelo in vitro para el estudio del ciclo biológico de L. chagasi y L. braziliensis.
The susceptibility of culture cells derived from embryonic tissues of Aedes aegypti to the infection with Leishmania (L) chagasi and Leishmania (V) braziliensis was evaluated. Methodology: These parasites are etiological agents of American visceral leishmaniasis and cutaneous leishmaniasis, respectively. Selected cells of Aedes aegypti were maintained in culture medium Grace/L15, supplement with 15% bovine fetal serum, 5,4 mg/ml of albendazol and an antibiotic mixture and incubated at an average temperature of 26°C. The cultures were inoculated with metacyclic promastigotes of the strain MH/ CO/84/CI-044B of L. chagasi and the strain HOM/BR752903 of L. braziliensis in a concentration of 10 parasites by cell. The J774 cell line was used as positive control of infection. Results: The highest percentage of infection represented as the number of amastigotes per cell in A. aegyti cell cultures and in the J774 cell line were obtained on days 6 and 9 post-infection. The results showed interaction, internalization and maturation in vitro of the two species of the parasite in the cells of a non-vector insect of Leishmania. Infected A. aegypti cells showed changes in its area because of the influence of the parasites that differ significantly (P <0.05) compared to not infected cells. Conclusion: Cell cultures from A. aegypti emerge as a new in vitro model for the study of the biological cycle of L. chagasi and L. braziliensis.
Subject(s)
Animals , Aedes , Virulence , Infections , LeishmaniaABSTRACT
The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6 percent) and on day 4 in the J774 cells (51 percent). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.
Subject(s)
Humans , Animals , Leishmania infantum/growth & development , Psychodidae/cytology , Cell Line/parasitology , Leishmania infantum/ultrastructure , Microscopy, Electron , Psychodidae/parasitologyABSTRACT
Introducción. Los cultivos celulares de insectos son una metodología útil en estudios biomédicos y tecnológicos.Objetivo. El propósito principal del presente trabajo fue obtener y caracterizar cultivos celulares derivados de tejidos embrionarios de Aedes aegypti. Materiales y métodos. Se emplearon huevos embrionados para los explantes de tejidos en los medios de cultivos MM/VP12 y L-15/Grace, con suplemento de 20 por ciento de suero fetal bovino y una mezcla al 1 por ciento de antibiótico y antimicótico, con un rango de pH entre 6,8 y 7,0. Los cultivos se incubaron a una temperatura de 28oC sin atmósfera de CO2. Resultados. El crecimiento celular se obtuvo en el medio L-15/Grace, 3 semanas después de haber sido sembrados los tejidos embrionarios; sin embargo, se necesitaron 6 meses para la formación de la monocapa confluente. Desde agosto de 2003 hasta junio de 2004, se habían realizado 28 subcultivos. Las células se caracterizaron morfológicamente; predominaron las formas epitelioides en subcultivos de pases altos. También se reconocieron las particularidades morfométricas del cariotipo y, además, se determinaron los perfiles isoenzimáticos y moleculares de los cultivos celulares, los cuales se compararon con muestras de adultos de la especie tomadas de la misma colonia y con líneas celulares derivadas de otros insectos.Discusión y conclusiones. Estas células representan, potencialmente, un importante sistema in vitro en investigaciones básicas y aplicadas
Subject(s)
Aedes/embryology , Cells, Cultured , In Vitro Techniques , Cytoplasmic Vesicles , Isoenzymes , Polymerase Chain Reaction , Random Amplified Polymorphic DNA TechniqueABSTRACT
Lutzomyia spinicrassa is a vector of Leishmania braziliensis in Colombia. This sand fly has a broad geographical distribution in Colombia and Venezuela and it is found mainly in coffee plantations. Baseline biological growth data of L. spinicrassa were obtained under experimental laboratory conditions. The development time from egg to adult ranged from 59 to 121 days, with 12.74 weeks in average. Based on cohorts of 100 females, horizontal life table was constructed. The following predictive parameters were obtained: net rate of reproduction (8.4 females per cohort female), generation time (12.74 weeks), intrinsic rate of population increase (0.17), and finite rate of population increment (1.18). The reproductive value for each class age of the cohort females was calculated. Vertical life tables were elaborated and mortality was described for the generation obtained of the field cohort. In addition, for two successive generations, additive variance and heritability for fecundity were estimated.
Subject(s)
Animals , Male , Female , Insect Vectors , Laboratories , Life Cycle Stages , Life Tables , Psychodidae , Population Dynamics , Quantitative Trait, Heritable , ReproductionABSTRACT
Con el propósito de establecer algunas características citogenéticas de cinco especies nativas de Lutzomyia, correspondientes a la serie townsendi del grupo verrucarum: Lutzomyia longiflocosa, Lutzomyia quasitownsendi, Lutzomyia spinicrassa, Lutzomyia torvida y Lutzomyia youngi, se llevó a cabo un estudio comparativo entre los cariotipos y su morfometría cromosómica. A partir de ganglios cerebrales de larvas de IV estadio, se prepararon los cromosomas mitóticos mediante la técnica de aplastamiento (squash) del tejido. Se efectuaron las mediciones cromosómicas, atendiendo los siguientes par metros: brazo corto, brazo largo, relación de brazos, longitud total, longitud relativa, índice centromérico y longitud relativa promedio. Se clasificaron los cromosomas según su morfometría y posición del centrómero siguiendo patrones estandarizados. Se calculó la distancia taxonómica y con base en estos datos se separaron las especies y se ubicaron en un fenograma. Las cinco especies de flebótomos presentaron 4 pares de cromosomas, número diploide (2N8), y el número fundamental fue de 16. En ninguno de los cariotipos se observó heteromorfismo sexual cromosómico. El análisis estadístico de los datos de morfometría cromosómica mostró diferencias altamente significativas entre los pares cromosómicos de las cinco especies. Sin embargo, la longitud total del genoma en los flebotómos fue muy similar, a excepción de L. youngi. En conclusión, las especies íntimamente relacionadas se lograron diferenciar a nivel citotaxonómico.
Subject(s)
Immunologic Deficiency Syndromes , Relapsing Fever , PhenotypeABSTRACT
A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20 percent fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10 percent fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma
Subject(s)
Animals , Arboviruses , Cell Line , Culicidae/genetics , Cell Line/chemistry , Cell Line/cytology , Cell Line/virology , Culicidae/virology , Time FactorsABSTRACT
The brain cell karyotype of New World sand fly Lutzomyia shannoni was described. This species has four pairs of chromosomes, 2N=8, with one pair of heteromorphic chromosomes
Subject(s)
Animals , Male , Female , Brain/cytology , Chromosome Banding , Psychodidae/genetics , Chromosome Banding , Karyotyping , MetaphaseABSTRACT
Con el propósito de comparar citogenéticamente dos cepas colombianas del mosquito Psorophora confinnis, procedentes de Lorica ( Córdoba) y Granada (Meta), se efectuó un estudio de caracterización cariólogica. A partir de tejidos cerebrales de larvas de IV estadio, se prepararon los cromosomas mitóticos mediante la técnica de aplastamiento (Squash) del tejido. Se efectuaron las mediciones cromosómicas, atendiendo a los siguientes parámetros: brazo corto, brazo largo, relación de brazos, longitud total, longitud relativa, índice centromérico y longitud relativa de los cromosomas. Se compararon los cálculos morfométricos y se aplicaron patrones estandarizados para clasificar los cromosomas en los cariotipos analizados. Se registraron diferencias altamente significativas en la longitud total cromosómica y también en los valores de los brazos cortos y largos de los cromosomas, en las metafases obtenidas de las dos cepas. Teniendo en cuenta los patrones de bandas C y G, se estableció correctamente la ubicación del centrómero y las regiones heterocromáticas asociadas a él e, igualmente, el número de bandas en cada uno de los pares cromosómicos en el cariotipo de las cepas de Lorica y Granada. Los resultados de los datos morfométricos y de los fenotipos de bandeamiento sugieren que Ps. confinnis existe en Colombia como un complejo de especies
Subject(s)
Animals , Culicidae/genetics , Karyotyping , Chromosome BandingABSTRACT
Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.