ABSTRACT
The concept of electronic orbitals has enabled the understanding of a wide range of physical and chemical properties of solids through the definition of, for example, chemical bonding between atoms. In the transmission electron microscope, which is one of the most used and powerful analytical tools for high-spatial-resolution analysis of solids, the accessible quantity is the local distribution of electronic states. However, the interpretation of electronic state maps at atomic resolution in terms of electronic orbitals is far from obvious, not always possible, and often remains a major hurdle preventing a better understanding of the properties of the system of interest. In this review, the current state of the art of the experimental aspects for electronic state mapping and its interpretation as electronic orbitals is presented, considering approaches that rely on elastic and inelastic scattering, in real and reciprocal spaces. This work goes beyond resolving spectral variations between adjacent atomic columns, as it aims at providing deeper information about, for example, the spatial or momentum distributions of the states involved. The advantages and disadvantages of existing experimental approaches are discussed, while the challenges to overcome and future perspectives are explored in an effort to establish the current state of knowledge in this field. The aims of this review are also to foster the interest of the scientific community and to trigger a global effort to further enhance the current analytical capabilities of transmission electron microscopy for chemical bonding and electronic structure analysis.
ABSTRACT
Resumen La leptospirosis continúa siendo hoy en día un problema para la salud pública, principalmente en poblaciones de bajos recursos socioeconómicos. En este trabajo se presenta la detección de leptospiras patógenas en muestras ambientales (aguas y barros) provenientes de regiones del norte argentino (provincias de Formosa, Salta, Santiago del Estero, Misiones y Chaco) con variadas características climatológicas habitadas por poblaciones vulnerables. De las 89 muestras analizadas, en el 24,7% fue posible detectar molecularmente la presencia de leptospiras patógenas. La prevalencia por tipo de muestra fue de 27,8% para las aguas y 11,8% para los barros. Todas las localidades muestreadas presentaron al menos una muestra positiva a alguna de las pruebas realizadas, por lo que el presente trabajo refleja la necesidad de profundizar los estudios de la leptospirosis en distintas regiones de la Argentina.
Abstract Leptospirosis remains as a major public health problem nowadays, mainly affecting vulnerable communities with low socioeconomic resources. In this study, the molecular detection of pathogenic leptospires from environmental samples (water and mud) from northern Argentina (Formosa, Salta, Santiago del Estero, Misiones and Chaco provinces) is described. Samples were obtained from regions with varied climatological features, all inhabited by vulnerable communities. From the 89 samples that were analyzed, 24.7% showed molecular evidence of the presence of pathogenic leptospires. Prevalence by sample type was: 27.8% in water samples and 11.8% in mud samples. All the sampled regions showed at least one positive sample. This result highlights the need of further research regarding leptospirosis in different regions of Argentina.
ABSTRACT
Abstract Molecular tools have improved conventional veterinary diagnosis. Acid nucleic extraction is a key step for downstream applications. This work aimed to compare the DNA extraction method Chelex-100 resin (M1) with Whatman® cards (M2), phenol-chloroform (M3), or commercial kits (M4), and to determine the most sensitive and inexpensive one for its diagnosis of animal pathogens that, despite their economic or zoonotic relevance, receive little attention. DNA was isolated from urine, organs, semen, blood and intestinal mucous, from the bacteria Leptospira interrogans serovar Pomona Pomona (by M1 and M2), Brucella melitensis (by M1, M3 and M4), and Salmonella ser. Abortusequi (by M1 and M4), and the parasites Leishmania spp. (by M1, M3 and M4), and Eimeria spp. (by M1 and M3), respectively. The sensitivity of each method was assayed by Polymerase Chain Reaction (PCR). The M1 showed similar sensitivity for Salmonella ser. Abortusequi, Leishmania spp., and Eimeria spp., being better for L. interrogans serovar Pomona Pomona and slightly lower for B. melitensis. For the first time, a simple and economic method was successfully employed for extracting DNA from these animal pathogens, especially important in low-resource settings, contributing to the diagnosis of leptospirosis, brucellosis, leishmaniasis, and coccidiosis; as well as to the molecular epidemiology of salmonellosis in stallion from semen samples.
Resumen Las técnicas moleculares han contribuido a mejorar el diagnóstico veterinario tradicional y la extracción de ácidos nucleicos es determinante. El objetivo de este trabajo fue comparar el método de extracción de ADN Chelex-100 (M1) con papel Whatman (M2), fenol-cloroformo (M3) o kits comerciales (M4), y determinar un método sensible y de bajo costo para el diagnóstico de patógenos de animales económica o zoonóticamente relevantes y que reciben poca atención. A partir de orina, órganos, semen, sangre y mucosa intestinal se extrajo el ADN de las bacterias Leptospira interrogans serovar Pomona Pomona (con M1 y M2), Brucella melitensis (con M1, M3 y M4), Salmonella ser. Abortusequi (M1 y M4), y de los parásitos Leishmania spp. (M1, M3 y M4) y Eimeria spp. (M1 y M3), respectivamente. La sensibilidad de los protocolos fue analizada por PCR. El método M1 demostró una sensibilidad similar para S. Abortusequi, Leishmania spp. y Eimeria spp., siendo mejor para L. interrogans y levemente menor para B. melitensis. Por primera vez se usó exitosamente en estos patógenos veterinarios un método simple y económico para extraer ADN, especialmente importante en laboratorios de bajos recursos económicos, contribuyendo al diagnóstico de leptospirosis, brucelosis, leishmaniasis y coccidiosis, así como también a la epidemiología molecular de salmonelosis en muestras de semen de caballos.
ABSTRACT
La leptospirosis bovina es una importante enfermedad zoonótica cuyo diagnóstico molecular está ampliamente divulgado. Sin embargo, no existe un método único de extracción de ADN para leptospiras patógenas a partir de muestras clínicas. En este trabajo se utilizó orina bovina contaminada con cultivo de L. interrogans serovar Pomona Pomona para analizar el mejor método comparando: M1.) resina Chelex-100, M2.) papel FTA Whatman y M3.) hervido de la muestra (protocolo casero). De estas tres técnicas, la primera (M1) presentó la mayor sensibilidad al realizar la PCR de diagnóstico, detectándose hasta 2x102 leptospiras/mL. La metodología aquí planteada resultó tener buen rendimiento para la detección de leptospiras en muestras clínicas animales, aunque es necesario su validación con mayor número y diversidad de muestras.
Bovine leptospirosis is an important zoonotic disease whose molecular diagnosis is widely reported. However, there is not a unique method of extraction of DNA for pathogenic leptospires using clinical samples. In this study, bovine urine was contaminated with pure culture of L. interrogans serovar Pomona Pomona in order to compare three of them: M1.) Chelex-100 resin, M2.) FTA Whatman paper and M3.) boiling of the sample (in-house protocol), being the first one the most sensitive when used in diagnostic PCR, detecting up to 2x102 leptospiras/mL. The methodology proposed in this study turned out to have good performance for the detection of leptospires in animal clinical samples, although it should be applied to a greater number of samples and in different stages of the pathology.
ABSTRACT
La leptospirosis es una zoonosis de amplia distribución mundial y de importancia en salud pública y veterinaria. El serogrupo Icterohaemorrhagiae toma importancia, ya que ha sido aislado con frecuencia en roedores y en casos clínicos de humanos, caninos, bovinos y porcinos; y se ha logrado aislar a partir de agua del rio Reconquista, provincia de Buenos Aires, Argentina. El objetivo de este estudio fue discriminar molecularmente entre las serovariedades Copenhageni e Icterohaemorrhagiae del serogrupo Icterohaemorrhagiae de importancia en Argentina, utilizando la técnica del análisis de repeticiones en tándem en múltiples locus (MLVA) y analizar las distancias génicas entre los genotipos determinados a partir de cepas aisladas de animales. Para ello, genotipificamos las 4 cepas referenciales del serogrupo Icterohaemorrhagiae de la especie Leptospira interrogans serovariedad Copenhageni cepa M20 y cepa Ictero I y de la serovariedad Copenhageni cepa RGA y cepa Fiocruz L1-130. Para este estudio se incluyeron 24 cepas aisladas de animales domésticos como silvestres. Logramos mediante esta técnica determinar un código numérico para cada serovariedad y así pudimos discriminar entre las serovariedades de este serogrupo. El análisis de coordenadas principales (PCoA) demostró la variabilidad génica existente entre las 4 cepas referenciales pertenecientes al serogrupo Icterohaemorrhagiae, el genotipo con mayor representación fue el serovar Copenhageni cepa Fiocruz L1-130 con un total de 13 cepas aisladas con ese perfil genético. Discriminar las serovariedades de este importante serogrupo nos permitirá en un futuro investigar si existen diferencias entre la sintomatología clínica y/o respuesta al tratamiento en los casos clínicos producidos por las distintas serovariedades del serogrupo Icterohaemorrhagiae discriminadas en este trabajo.
Leptospirosis is the world`s most widely distributed zoonosis and of great importance in animal health and in public health. The relevance of the serogroup Icterohaemorrhagiae relies on the frequent isolation from rodents and from clinical cases of humans, canines, bovines and swine; and this serogroup was also isolated from water samples of the Reconquista river, Buenos Aires province, Argentina. The objective of this study was to discriminate molecularly between serovars Copenhageni and Icterohaemorrhagiae being of importance in Argentina, using Multiple Locus Variable number tandem repeats analysis (MLVA) and analyzing the genetic distances among genotypes obtained from animal isolates in this study. We genotyped the four referential strains of the serogroup Icterohaemorrhagiae belonging to the species Leptospira interrogans serovar Copenhageni strain M20 and strain Ictero I and of the serovar Copenhageni strain RGA and strain Fiocruz L1-130. In this study we included 24 strains isolated from animals domestic and wildlife. Using this technique we could determine numeric codes for each serovar and in this way, discriminate between serovars of this serogroup. The principal coordinates analysis (PCoA) showed the genetic variability of the four referential strains from the serogroup Icterohaemorrhagiae, the genotype with better representation was serovar Copenhageni strain Fiocruz L1-130 with 13 isolated strains with this genetic profile. Discrimination of the serovars from this important serogroup allows to establish differences between clinical signs and/or response to treatment in the clinical cases produced by different serovars of the serogroup Icterohaemorrhagiae successfully distinguished in this study.