ABSTRACT
Intestinal pathogenic microorganisms are introduced into the water by means of faecal contamination, thus creating a threat to public health and to the environment. Detecting these contaminants has been difficult due to such an analysis being costly and time-intensive; as an alternative, microbiological indicators have been used for this purpose, although they cannot differentiate between human or animal sources of contamination because these indicators are part of the digestive tracts of both. To identify the sources of faecal pollution, the use of chemical, microbiological and molecular markers has been proposed. Currently available markers present some geographical specificity. The aim of this study was to select microbial and molecular markers that could be used to differentiate the sources of faecal pollution in the Bogotá River and to use them as tools for the evaluation and identification of the origin of discharges and for quality control of the water. In addition to existing microbial source markers, a phage host strain (PZ8) that differentiates porcine contamination was isolated from porcine intestinal content. The strain was identified biochemically and genotypically as Bacteroides. The use of this strain as a microbial source tracking indicator was evaluated in bovine and porcine slaughterhouse wastewaters, raw municipal wastewaters and the Bogotá River. The results obtained indicate that the selected microbial and molecular markers enable the determination of the source of faecal contamination in the Bogotá River by using different algorithms to develop prediction models.
Subject(s)
Bacteroides/isolation & purification , Environmental Monitoring/methods , Feces , Water Pollutants/isolation & purification , Water Pollution , Abattoirs , Animals , Cattle , Colombia , Machine Learning , Rivers/microbiology , Swine , Wastewater/microbiology , Water MicrobiologyABSTRACT
The microbiological indicators traditionally used to assess fecal contamination are insufficient to identify the source. The aim of this study was to detect microbial markers to identify the source of fecal pollution in the Bogotá River (Colombia). For this, we determined non-discriminating indicators such as Escherichia coli, somatic coliphages and phages infecting strain RYC2056 of Bacteroides, and potential source tracking markers as phages infecting strains GA17, HB13, and CA8 of Bacteroides, sorbitol-fermenting bifidobacteria, and molecular markers of Bifidobacterium adolescentis, Bifiodobacterium dentium, and Bacteroidetes in raw municipal wastewaters, slaughterhouse wastewaters, and the Bogotá River. Bacteriophages infecting Bacteroides strain GA17 and the molecular markers identified the wastewater sources. In contrast, sorbitol-fermenting bifidobacteria failed regarding specificity. In the Bogotá River, phages infecting strain GA17 were detected in all samples downstream of Bogotá, whereas they should be concentrated from 1 l samples in upstream samples containing less than 10(3) E. coli/100 ml to be detected. In the river water, the fraction of positive detections of molecular markers was lower than that of phages infecting strain GA17. The ratio SOMCPH/GA17PH was shown also to be a good marker. These results provide information that will allow focusing measures for sanitation of the Bogotá River.
Subject(s)
Bacteroides/genetics , Genetic Markers/genetics , Abattoirs , Colombia , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Rivers/microbiology , Wastewater/microbiologyABSTRACT
Trypanosoma cruzi kinetoplastid membrane protein-11 (KMP-11) is able to induce protective immunity in mice. In humans, T-cell immunity during Chagas disease has been documented using parasite antigens allowing the identification of specific CD8(+) T cells. However, little is known about the CD4(+) T-cell response during the evolution of the disease. In this paper, the induction of a natural CD4(+) T-cell response against the KMP-11 protein in T. cruzi infected humans was studied to assess whether this parasite-derived protein could be processed, presented and detected as a major histocompatibility complex class II restricted epitope. The results show that helper T cells from 5 out of 13 chagasic patients specifically produced interferon-gamma after exposure to the KMP-11 antigen, whereas healthy donors and non-chagasic cardiopathic patients did not respond. This is the first description of T. cruzi KMP-11 protein recognition by CD4(+) T cells in chronic chagasic patients.
Subject(s)
Biomarkers/blood , CD4-Positive T-Lymphocytes/immunology , Chagas Disease/immunology , Protozoan Proteins/blood , Protozoan Proteins/immunology , Trypanosoma cruzi/immunology , Animals , Biomarkers/metabolism , Chagas Disease/physiopathology , Cloning, Molecular , Female , Humans , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Male , Mice , Middle Aged , Protozoan Proteins/geneticsABSTRACT
La aplicación de la reacción en cadena de la polimerasa (PCR) para detectar e identificar Trypanosoma rangeli y Trypanosoma rangeli presenta a menudo dificultades de interpretación. Así, algunas pruebas generan la amplificación de bandas similares provenientes de uno de los dos parásitos, fragmentos polimórficos de un mismo parásito, o la prevalencia en la detección de T. cruzi en infecciones mixtas. En este estudio se presentan y analizan los trabajos de investigación básica realizados con el objeto de diseñar y estandarizar pruebas de PCR específicas de cada parásito. Los iniciadores TcH2AF/R se diseñaron sobre la base de la región diferencial observada entre las unidades génicas que contienen los genes h2a en estos tripanosomas. Esta pareja de iniciadores amplifican un fragmento de 234 pb específico para T. cruzi (cepas I y II). Los iniciadores TrF/R2 anillan en las regiones intergénicas del fragmento génico de 801 pb codificante para seis transcritos que forman la agrupación ARNsno-Cl en T. rangeli. Estos iniciadores amplifican un fragmento de 620 pb exclusivo de las cepas KP1(-) y KP1(+) de este parásito. La aplicación de estas PCR en vectores infectados y en pacientes con enfermedad de Chagas muestra que ambas pruebas constituyen herramientas útiles para el diagnóstico y la identificación diferencial de estos tripanosomátidos.
The application of polymerase chain reaction (PCR) to detect Trypanosoma rangeli and Trypanosoma rangeli often presents interpretation challenges. For example, some tests yield the amplification of similar bands from either parasite, polymorphic fragments of the same parasite, or present deviation towards T. cruzi in mixed infections. In this study, the basic researching needed for designing and standardizating specific PCR tests for each parasite species PCR are shown and analyzed. The TcH2AF/R primers were designed on the basis of the differential gene region observed between the histone h2a genic units of these parasites. These primers amplify a specific 234 bp fragment in T. cruzi (T. cruzi I and II strains). The TrF/R2 primers anneal to the intergenic regions of an 801 bp gene fragment encoding for six transcripts that conform the snoRNA-Cl cluster in T. rangeli. These primers amplify a fragment of 620 bp exclusively in KP1(-) and KP1(+) strains of the parasite. The application of these PCR tests in infected vectors and in chagasic patients show that both tests constitute useful tools for the diagnosis and differential identification of these Trypanosomatids. Key words: histone, RNA small nucleolar (snoRNA), polymerase chain reaction (PCR), Trypanosoma.
Subject(s)
RNA, Small Nuclear , Histones , Diagnostic Tests, Routine , Polymerase Chain Reaction , Trypanosoma , ColombiaABSTRACT
We describe the localization of the KMP-11 protein in the Trypanosoma rangeli parasite determined by immunoelectron microscopy using a monoclonal antibody generated against the Trypanosoma cruzi KMP-11 protein. The data reported herein show that the T. rangeli KMP-11 protein is mainly accumulated in the parasite cytoplasm, the coat, the flagellum, and the flagellar pocket. The high degree of sequence homology between the KMP-11 proteins from both parasites suggests that the KMP-11 protein from T. rangeli, like that of T. cruzi, could also be associated with the parasite cytoskeleton.
Subject(s)
Antibodies, Monoclonal/immunology , Cytoskeleton/immunology , Membrane Glycoproteins/immunology , Protozoan Proteins/immunology , Trypanosoma/metabolism , Animals , Cytoskeletal Proteins , Flagella , Membrane Glycoproteins/analysis , Microscopy, Immunoelectron/methods , Protozoan Proteins/analysisABSTRACT
Genes encoding for the KMP-11 protein were localized on the chromosomes of Trypanosoma rangeli. These genes were located in two chromosomes of 3,100 and 3,400 kb in the KP1(-) strain whereas in the KP1(+) H14 and Choachí strains, the genes are located in a chromosome of 1,600 kb. The Choachí strain presents an additional band of 1,400 kb. In the Shubacbarina and Munanta strains of Trypanosoma cruzi, the KMP-11 genes are located on a chromosomal band of 1,490 kb. Therefore, the chromosomal localization of the KMP-11 genes presents a potential tool to differentiate among these parasites.
Subject(s)
Membrane Glycoproteins/genetics , Protozoan Proteins/genetics , Trypanosoma/genetics , Animals , Chromosomes, Bacterial/geneticsABSTRACT
Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is a public health problem in Colombia. Previous reports have indicated the presence of heterogeneity among parasite populations. Six Colombian T. cruzi strains were obtained that differed by host, geographical region and transmission cycle. The genetic variability of each was compared by random amplified polymorphic DNA (RAPD), and isoenzymes. A restriction fragment length polymorphism (RFLP) was extracted using the 1.2 kb unit encoding the parasite's H2A histone as a probe. Genetic distances between the isolates varied greatly, from 0.611 to 0.99 as determined by RAPD profiles (M13F and M13R primers), between 0 and 0.81 by RFLP profiles (5 endonucleases), and between 0.10 and 0.55 by isoenzymes (13 enzymatic systems). Genetic distance matrixes derived from each of the three methods showed that Colombian strains exhibit a high degree of genetic differentiation. This may account for the broad clinical spectrum of Chagas disease in Colombia.
Subject(s)
Nucleic Acid Amplification Techniques , Polymorphism, Restriction Fragment Length , Trypanosoma cruzi/genetics , Animals , Colombia , Trypanosoma cruzi/classificationABSTRACT
Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is a public health problem in Colombia. Previous reports have indicated the presence of heterogeneity among parasite populations. Six Colombian T. cruzi strains were obtained that differed by host, geographical region and transmission cycle. The genetic variability of each was compared by random amplified polymorphic DNA (RAPD), and isoenzymes. A restriction fragment length polymorphism (RFLP) was extracted using the 1.2 kb unit encoding the parasite's H2A histone as a probe. Genetic distances between the isolates varied greatly, from 0.611 to 0.99 as determined by RAPD profiles (M13F and M13R primers), between 0 and 0.81 by RFLP profiles (5 endonucleases), and between 0.10 and 0.55 by isoenzymes (13 enzymatic systems). Genetic distance matrixes derived from each of the three methods showed that Colombian strains exhibit a high degree of genetic differentiation. This may account for the broad clinical spectrum of Chagas disease in Colombia.
Subject(s)
Animals , Nucleic Acid Amplification Techniques , Polymorphism, Restriction Fragment Length , Trypanosoma cruzi , Colombia , Trypanosoma cruziABSTRACT
In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.
Subject(s)
Chagas Disease/diagnosis , DNA, Protozoan/isolation & purification , Polymerase Chain Reaction/standards , RNA, Small Nucleolar/genetics , Trypanosoma cruzi/isolation & purification , Trypanosoma/genetics , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Chagas Disease/parasitology , DNA Primers , DNA, Protozoan/chemistry , Diagnosis, Differential , Gene Library , Insect Vectors/parasitology , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Polymerase Chain Reaction/methods , RNA, Protozoan/genetics , Rhodnius/parasitology , Sequence Alignment , Species Specificity , Trypanosoma/isolation & purification , Trypanosoma cruzi/geneticsABSTRACT
El presente trabajo presenta la descripcion de un proyecto de un Sistema de Supervision, control de energia y seguridad en condominios. Se refiere a la supervision y control a traves de sistemas integrados con la finalidad de obtener: -Control centralizado de operacion de equipos y sistemas de control de los diferentes puntos a ser controlados -Optimizacion operacional y de mantenimiento -Racionalizacion de energia. En el desarrollo del proyecto se pretende un resumen sobre tecnicas de supervision y control que pueden ser aplicados en edificios y condominios. Se formularan los problemas de control y operacion de edificios, ademas se describe los tipos de puntos remotos comunmente encontrados en un Sistema de Supervision, control de energia y seguridad en condominios. Se describira un proyecto basico de un sistema de supervision, control de energia y seguridad en condominios, el cual, es considerado para ser implementado en la Cooperativa de Telefonos Auotmaticos Cotas Ltda. de la ciudad de Santa Cruz, o sea, se dara una vision global del sistema, con la intencion de presentar una especificacion de un sistema de supervision, control de energia y seguridad en condominios. esta especificacion no pretende abarcar todos los asuntos relativos a la supervision y control de condominios, solo se trata de presentar una propuesta de solucion para los principales problemas. son abarcados las laternativas de configuracion de un sistema de Supervision, Control de energia y seguridad en Condominios, tambien los requisitos basicos para tal configuracion. son abordados tambien, las funciones minimas a ser efectuadas por un sistema de supervision, control de energia y seguridad en condominios y la interfase homnre-maquina necesaria. El proyecto de software para la estacion central es descrito enlenguaje corriente, no siendo usado ningun tipo de lenguaje de programacion, no implicando ninguna restriccion al proyecto. El software de la estacion remota fue escrito en lenguaje de maquina assembler del microprocesador Z-80.
