Abstract
Morphology and cytochemistry of Aedes aegypti's cell cultures (Diptera: Culicidae) and susceptibility to Leishmania panamensis (Kinetoplastida: Trypanosomatidae). The first cellular line of Aedes aegypti was developed by Grace in 1966; afterwards, other cellular lines of this species have been generated. These have been used for the study of pathogenic organisms like viruses, bacteria and parasites, which demonstrates their importance in biomedical applications. This research describes, for the first time, some cytochemical characteristics of A. aegypti cell cultures, that were infected with (MHOM/CO/87CL412) strain of Leishmania panamensis. A morphological study of the cell culture was also carried out. Maintenance of the cell culture, parasites and infection in vitro were carried out in the Laboratory of Entomology, Cell Biology and Genetics of the Universidad de La Salle. The cell cultures infected with the parasite were maintained in a mixture of mediums Grace/L15, supplemented with 10 % fetal bovine serum (FBS) at pH 6.8 and a temperature of 26 degrees C, during 3, 6 and 9 post-infection days. After this, these cell cultures were processed through High Resolution Light Microscopy (HRLM) and Transmission Electron Microscopy (TEM) based on standard protocols defined by the Group of Microscopy and Image Analyses of the Instituto Nacional de Salud. Semi-fine slices of 1 microm colored with toluidine blue were used for the morphological analysis of the culture, and ultra fine cuts of 60 to 90 nm stained with uranyl acetate and lead citrate where used for the ultrastructural study. In addition, PAS and peroxidase staining was carried out in cells fixed with methanol. The morphometric study was analyzed with software ImageJ (NIH). In the semi-fine slices, small cells were observed showing fibroblastic appearance 10.84 +/- 2.54 microm in length and 5.31 +/- 1.26 microm wide; other cells had epithelial appearance with a great peripheral nucleus, voluminous and vacuolated cytoplasm, 23.04 +/- 4.00 microm in length and 13.96 3.70 microm wide. These last ones predominated over the ones with fibroblastic appearance. Regarding the PAS coloration, 7.08% of the cells presented abundant PAS positive cytoplasmatic granules which indicated polysaccharides presence. The peroxidase test gave a negative result. The greatest percentage of infection (18.90%) of one total of 101 cells, turned up by day 6. Some cells analyzed by TEM presented a vacuolated aspect cytoplasm; some contained parasites, other fibrillar material and others were empty. The results indicate that A. aegypti cell culture can support the internalization and transformation of the parasite, which demonstrates the capacity that these cell cultures have to be infected with L. panamensis and to maintain the infection for approximately one week.
Highlights
Desde comienzos del siglo XX los entomólogos han tratado de mantener in vitro células de insecto como una herramienta en diferentes campos de estudio (Lynn 2002)
Con lo descrito en los anteriores antecedentes, se pretende resaltar la importancia que tienen los cultivos celulares de mosquitos, en el mantenimiento y estudio del ciclo biológico de tripanosomátidos, importantes en salud publica
La observación de las muestras fueron realizadas en un microscopio de luz Zeiss Axiophot
Summary
Cultivos celulares e infección: El mantenimiento de los cultivos celulares, parásitos y la infección in vitro, fueron realizados en el Laboratorio de Entomología, Biología Celular y Genética de la Universidad de La Salle. Los cultivos celulares de A. aegypti fueron sembrados en seis placas de petri de 35x10 mm e infectados con la cepa (MHOM/CO/87CL412) de L. panamensis, suministrada por el laboratorio de parasitología del Instituto Nacional de Salud (INS). La observación de las muestras fueron realizadas en un microscopio de luz Zeiss Axiophot. Morfometría celular: La captura de imágenes de las células en los días 3, 6 y 9 post-infección y de las células no infectadas, se realizó por medio de una cámara Sony Hyper HAD CCD-IRIS/RGB adaptada a un microscopio de luz Zeiss Axiophot. Morfología: En los cultivos celulares correspondientes a células no infectadas, se observaron dos grupos de células morfológicamente diferentes: uno constituido por células de apariencia fibroblastoide con unas dimensiones de 10.84±2.54 μm de largo y 5.31±1.26 μm de ancho, cuyo citoplasma presentaba algunas vacuolas, como se observa en la Fig. 1. El Cuadro 1 nos muestra el promedio del tamaño celular (longitud y amplitud) de las dos morfologías celulares encontradas
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
