Metagenomics and Diagnosis of Zoonotic Diseases

Zoonotic diseases represent a public health problem worldwide, since approximately 60% of human pathogens have a zoonotic origin. A variety of methodologies have been developed to diagnose zoonosis, including culture-dependent and immunological-based methods, which allow the identification of a huge range of pathogens. However, some of them are not detected easily with these approaches. Additionally, molecular tests have been developed, and they are designed to identify a single pathogen or mixtures of them. In this context, metagenomics comes as an alternative to get genome sequences of different microorganisms, which comprise a microbial community. Metagenomics have been used to characterize microbiomes and viromes, which are not cultivable under laboratory conditions. This methodology could be a powerful tool in the diagnosis of zoonotic diseases because it allows not only identification of genus and species, but also detection of some proteins in specific conditions on specific tissues, through structural and functional metagenomics, respectively

Actividad biológica de Bacillus thuringiensis sobre la polilla guatemalteca de la papa, Tecia solanivora Povolny (Lepidoptera: Gelechiidae)

The potato (Solanum tuberosum) is one of the most important crops in Colombia . Larvae of the Guatemalan potato moth Tecia solanivora Povolny (Lepidoptera: Gelechiidae) cause direct damage to the tubers, producing economic losses and increased use of agrochemicals. Bacillus thuringiensis (Bt) is an alternative in the management of insect pests due to its specificity. Their activity depends on proteins known Cry, which when ingested by a susceptible insect form pores in their intestinal cells that cause death of the insect. This review presents studies on the effect of Bt to T. solanivora. It has been found that the Cry1Ac (basis for transgenic varieties) and Cry1B toxins have significant toxic activity. Also the experience of the design and evaluation of a hybrid toxin ( Cry1B - Cry1I ) which resulted in a significant lethality towar T. solanivora.La papa (Solanum tuberosum) es uno de los cultivos más importantes de Colombia. Las larvas de la polilla guatemalteca de la papa, Tecia solanivora Povolny (Lepidoptera: Gelechiidae), causan daños directos a los tubérculos, produciendo pérdidas económicas e incremento en el uso de agroquímicos. Bacillus thuringiensis (Bt) es una alternativa en el manejo de insectos plaga gracias a su especificidad. Su actividad depende de proteínas denominadas Cry, que cuando son ingeridas por un insecto susceptible forman poros en sus células intestinales que producen la muerte del insecto. Esta revisión presenta los estudios sobre el efecto de Bt hacia T. solanivora. Se ha encontrado que las toxinas Cry1Ac (base para variedades transgénicas) y Cry1B tienen importante actividad tóxica. Igualmente se destaca la experiencia del diseño y evaluación de una toxina híbrida (Cry1B-Cry1I) que resultó en una importante letalidad hacia T. solanivora

PRESERVACIÓN MODIFICADA Y DESCRIPCIÓN DE LA FUNGALISINA PARA BATRACHOCHYTRIUM DENDROBATIDIS

Batrachochytrium dendrobatidis es un hongo patógeno que causa quitridiomicosis, una afección cutánea que resulta en la reducción de las poblaciones de anuros en todo el mundo, debido a las fungalisinas, su factor de virulencia más importante. Los mapas de B. dendrobatidis (http://www.bd-maps.net/) contienen la información sobre los aislamientos relacionados con B. dendrobatidis. Una alternativa disponible para la reconstitución de cepas de B. dendrobatidis es a partir de células criopreservadas, los métodos para esto se basan en el protocolo de la Organización Mundial de Sanidad Animal (OIE). Recientemente, gran parte del interés en la investigación de B. dendrobatidis se ha centrado en la secuenciación de su ADN, se encuentran disponibles los genomas de B. dendrobatidis JAM81 y B. dendrobatidis JEL423. OBJETIVO. Evaluar una forma modificada del protocolo de la OIE para la criopreservación de cepas de B. dendrobatidis y el análisis in silico de su fungalisina. Se modificó el protocolo de la OIE utilizando la cepa de B. dendrobatidis EV001, enfocado en los ítems de concentraciones de criopreservantes, antibióticos y recuperación, desde 80 C, y evaluación de viabilidad microscópica en agar hidrolizado con triptona y gelatina. Además se utilizó Bioinformática para la determinación de características bioquímicas de la fungalisina de la cepa de B. dendrobatidis JAM81. El protocolo modificado de la OIE resultó útil para la recuperación de la viabilidad de la cepa B. dendrobatidis EV001. La caracterización de la fungalisina de B. dendrobatidis JAM81 mostró un peso molecular de 85 kDa, punto isoeléctrico de 8,33 y estructura terciaria característica que indicó que la proteína es una metaloproteinasa, tiene un dominio PepSY para la inhibición de proteasas, y un dominio catalítico asociado a destrucción de barreras proteicas./Batrachochytrium dendrobatidis is a pathogenic fungus causing chytridiomycosis, a cutaneous affection resulting in reduction on Anura populations around the world, because of fungalysins, the most important virulence factor. The B. dendrobatidis maps (http://www.bd-maps.net/) is the dataset of information about the B. dendrobatidis -related isolates. An alternative available for reconstitution of B. dendrobatidis strains from cryopreserved cells include methods relying on theWorld Organization for Animal Health (OIE) protocol. Most recently, much of the interest in the B. dendrobatidis research has focused on its DNA sequencing, especially B. dendrobatidis JAM81 and B. dendrobatidis JEL423 genomes. OBJECTIVE. To evaluate a modified form from OIE protocol for B. dendrobatidis strain criopreservation, and in silico analysis of B. dendrobatidis fungalysin. OIE protocol was modified using B. dendrobatidis EV001, focused on cryopreservant concentrations, antibiotics and recovering to 80 C, and microscopic viability evaluation on Tryptone Gelatin hydrolyzed agar. Besides, Bioinformatics was used for the determination of biochemical characteristics from a B. dendrobatidis JAM81 fungalysin. Modified OIE protocol was useful by viability, recovering B. dendrobatidis EV001 strain. The characterization of B. dendrobatidis JAM81 fungalysin showed a molecular weight of 85 kDa, isoelectric point of 8.33, and tertiary structure among others. This indicated that the protein is a metalloproteinase, it has a PepSY domain for protease inhibition, and a catalytic domain that destroy protein barriers

Actividad biológica de proteínas Cry recombinantes de Bacillus thuringiensis sobre larvas de primer instar de Hypothenemus hampei Ferrari (Coleóptera: Scolytidae)

INTRODUCCIÓN: La biología de la conservación propone aplicaciones para evitar la pérdida de biodiversidad. La agricultura de conservación es un modelo de producción agrario que ahorra en recursos para alcanzar beneficios satisfactorios, producción alta sostenida, y la protección del medio ambiente. El Manejo Integrado de Plagas es un plan fundamental en la agricultura de conservación que armoniza los métodos químicos y biológicos afín de proteger los ecosistemas agrícolas y sus recursos naturales (agua, suelo, aire). Hypothenemus hampei Ferrari (Coleóptera: Scolytidae) es el problema entomológico más importante en los cultivos de café del mundo. Se ha estimado que en Colombia se cultivan 800000 hectáreas de café, y esta plaga causa un gran perjuicio (pérdidas 8-50%). Se utilizan estrategias de control de esta plaga tales como técnicas culturales, enemigos naturales, entomopatógenos y agentes químicos. Bacillus thuringiensis (Bt) es una bacteria entomopatógena ampliamente utilizada en el control de insectos-plaga de los ordenes Lepidóptera, Coleóptera y Díptera. El factor de virulencia de Bt más importante son las proteínas Cry. Varias proteínas Cry tienen actividad letal contra los insectos coleópteros como Cry1B, Cry1I, Cry3 y Cry9. Se ha encontrado que cepas de Bt tipo israelensis son activas contra coleópteros. OBJETIVO: Evaluar la actividad biológica de proteínas Cry contra larvas de primer instar de H. hampei. RESULTADOS: Se construyeron las proteínas recombinantes Cry1B, Cry1I, Cry3A, Cry4, Cry9 y la proteína híbrida SN1917 (dominio II es una fusión de los correspondientes de Cry1I y Cry1B). Los bioensayos mostraron que Cry1B (mortalidad 43,5%) tiene un poco más de actividad que Cry3A (mortalidad 30,2%) contra el insecto. Desafortunadamente Cry1I, Cry4, Cry9 y SN1917 no poseen actividad hacia H. hampei. Las pruebas de western blot indican reconocimiento entre Cry1B, Cry1I o Cry3A y una proteína (140 kDa) de la membrana apical del epitelio del intestino medio del insecto. Un enfoque bioinformático señala la posible presencia de toxinas tipo Cry en genomas microbianos y ambientales con potencial contra coleópteros como H. hampei. CONCLUSIÓN: Se logro identificar proteínas Cry con potencial para el control biológico de larvas de la broca del café. / Abstract. INTRODUCTION: Conservation biology propose applications to avoid biodiversity loss. Conservation agriculture is a model for resource-saving farming production to reach satisfactory profits together, high and sustained production and environmental protection. Integrated Pest Management is a key plan in conservation agriculture to pest control that joins chemical and biological methods to protect crop ecosystem and surrounding natural resources (water, soil, air). Hypothenemus hampei Ferrari (Coleóptera: Scolytidae) is the most important entomological problem in coffee crops around the world. It has been estimated that 800000 Ha of coffee are cultivated in Colombia, and this pest causes an important damage (losses 8-50%). Several management strategies are used in their control such as cultural techniques, natural enemies, entomopathogens and chemical agents. Bacillus thuringiensis (Bt) is an entomopathogenic bacteria used widely in pest insect control of Lepidoptera, Coleoptera and Diptera orders. The most important virulence factor of Bt are Cry proteins. Several Cry proteins have lethal activity against coleopteran insects such as Cry1B, Cry1I, Cry3 and Cry9. It has been found that Bt israelensis-like strains are active against coleopterans. OBJECTIVE: To evaluate the biological activity of Cry proteins against first instar larvae of H. hampei. RESULTS: Recombinant Cry1B, Cry1I, Cry3A, Cry4, Cry9 and Hybrid protein SN1917 (domain II is a combination of corresponding Cry1I and Cry1B) were constructed. Bioassays showed that Cry1B (mortality 43.5%) is somewhat more active than Cry3A (mortality 30.2%) against the pest. Unfortunately, Cry1I, Cry4, Cry9 and SN1917 not possess activity on H. hampei. Binding tests indicate an interaction between Cry1B, Cry1I or Cry3A and a 140 kDa protein from apical membrane of epithelial midgut of this insect. A bioinformatics approach could be a sign of Cry-like toxins presence in microbial and environmental genomes with potential against Coleoptera order, such as H. hampei. CONCLUSION: In this work, it was possible to identify Cry proteins with potential to CBB biological control.Doctorad

Proteínas Cry de Bacillus thuringiensis y su interacción con coleópteros

Integrated Pest Management is a key plan in conservation agriculture to pest control that joins chemical and biological methods to protect crop ecosystem and surrounding natural resources (water, soil, air). Coleoptera order is an important entomological problem in commercial crops causing important losses at least up 15%. Several management strategies are used in their control such as cultural techniques, natural enemies, entomopathogens and chemical agents. Bacillus thuringiensis (Bt) is an entomopathogenic bacteria used widely in pest insect control of Lepidoptera, Coleoptera and Diptera orders. The most important virulence factor of Bt are Cry proteins. Several Cry proteins have lethal activity against coleopteran insects such as Cry1B, Cry1I, Cry3, Cry7, Cry8, Cry9, Cry14, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43 y Cry55. It has been found that Bt israelensis-like strains are active against coleopterans. In this review we show the most important determinants with relationship to biological activity of Bt against coleopteran-pestsEl Manejo Integrado de Plagas es un plan fundamental en la agricultura de conservación que armoniza los métodos químicos y biológicos afín de proteger los ecosistemas agrícolas y sus recursos naturales (agua, suelo, aire). El orden Coleóptera es uno de los problemas entomológicos más importante en los cultivos comerciales causando pérdidas de hasta 15%. Se utilizan estrategias de control de insectos tales como técnicas culturales, enemigos naturales, entomopatógenos y agentes químicos. Bacillus thuringiensis (Bt) es una bacteria entomopatógena ampliamente utilizada en el control de insectosplaga de los ordenes Lepidóptera, Coleóptera y Díptera. El factor de virulencia de Bt más importante son las proteínas Cry. Varias proteínas Cry tienen actividad letal contra los insectos coleópteros como Cry1B, Cry1I, Cry3, Cry7, Cry8, Cry9, Cry14, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43 y Cry55. Se ha encontrado que cepas de Bt tipo israelensis son activas contra coleópteros. En esta revisión nosotros presentamos los principales ítems relacionados con la actividad biológica de Bt sobre coleópteros-plaga

Actividad biológica de Bacillus thuringiensis sobre la polilla guatemalteca de la papa, Tecia solanivora Povolny (Lepidoptera: Gelechiidae)

La papa (Solanum tuberosum) es uno de los cultivos más importantes de Colombia. Las larvas de la polilla guatemalteca de la papa, Tecia solanivora Povolny (Lepidoptera: Gelechiidae), causan daños directos a los tubérculos, produciendo pérdidas económicas e incremento en el uso de agroquímicos. Bacillus thuringiensis (Bt) es una alternativa en el manejo de insectos plaga gracias a su especificidad. Su actividad depende de proteínas denominadas Cry, que cuando son ingeridas por un insecto susceptible forman poros en sus células intestinales que producen la muerte del insecto. Esta revisión presenta los estudios sobre el efecto de Bt hacia T. solanivora. Se ha encontrado que las toxinas Cry1Ac (base para variedades transgénicas) y Cry1B tienen importante actividad tóxica. Igualmente se destaca la experiencia del diseño y evaluación de una toxina híbrida (Cry1B-Cry1I) que resultó en una importante letalidad hacia T. solanivora

Evidence of gene flow between transgenic and non-transgenic maize in Colombia

Maize (Zea mays) is an important crop worldwide and is essential for industry. Many transgenic cultivars of maize have been developed over the years from this species, producing cultivars resistant to herbicides and insects, among other things. However, little is known about the gene flow processes that affect maize fields in Colombia, which is near the center of diversity for cultivated maize. We analyzed the gene flow phenomenon of 60 randomly chosen plots of maize, including farmer field landraces or other conventional varieties such as non-transgenic hybrids in Valle de San Juan (Colombia) using Inmunostrip®, PCR and ELISA tests on leaves (seed gene flow) and seeds (pollen gene flow). More than 88% of the plots were positive with the Inmunostrip® and PCR tests (35S promoter, Nos terminator and cry1F gene), using the leaves, while the remaining seven plots (12%) were positive for transgenic sequences in the seeds. The results indicated a significant level of overall transgene existence, which is consistent with gene flow from transgenic events. All of the field types (conventional maize, buffer zones, refuge, and Colombian landraces) showed evidence of a transgene presence. There are many problems that could increase the gene flow potential in Valle de San Juan, such as little respect for regulations (Colombian Decree 4525 on transgenic crops and biosafety), distance between transgenic and non-transgenic maize or use of refuge and/or buffer zones, high seed reuse and exchange and low technical assistance. Every policy decision must be made in light of scientific standards of judgment

La biotecnología de Bacillus thuringiensis en la agricultura

Bacillus thuringiensis (Bt) is a Gram-positive rod-shape bacterium that during its sporulation phase produces a parasporal inclusion formed by Cry proteins having biological activity against pest insects. Because of these proteins, Bt shows toxicity toward pest insect larvae of Lepidoptera, Co-leoptera and Diptera orders among others, besides of friendly with the environment, and for this reason Bt has a very common use for commercial products and transgenic plants with Cry protein basis in agriculture. In this review is described the most important Bt factors and their application as biotechnological tool in agrobusiness, considering Bt bioassays.Bacillus thuringiensis es un bacilo Gram positivo que durante su fase de esporulación produce una inclusión parasporal, conformada por proteínas Cry con actividad biológica contra insectos-plaga. Gracias a estas proteínas Bacillus thuringiensis presenta toxicidad contra larvas de insectos-plaga de los órdenes Lepidóptera, Coleóptera y Díptera, entre otros. Además es amigable con el medioambiente, razones por la cuales se ha hecho común el uso y desarrollo de productos comerciales y plantas transgénicas a base de toxinas Cry en el sector agrícola. En esta revisión se describirá los aspectos más importantes de Bt y su aplicación como herramienta biotecnológica para el sector agrícola, con una consideración sobre bioensayos usando esta bacteria

Evidence of gene flow between transgenic and non-transgenic maize in Colombia

Maize (Zea mays) is an important crop worldwide and is essential for industry. Many transgenic cultivars of maize have been developed over the years from this species, producing cultivars resistant to herbicides and insects, among other things. However, little is known about the gene flow processes that affect maize fields in Colombia, which is near the center of diversity for cultivated maize. We analyzed the gene flow phenomenon of 60 randomly chosen plots of maize, including farmer field landraces or other conventional varieties such as non-transgenic hybrids in Valle de San Juan (Colombia) using Inmunostrip®, PCR and ELISA tests on leaves (seed gene flow) and seeds (pollen gene flow). More than 88% of the plots were positive with the Inmunostrip® and PCR tests (35S promoter, Nos terminator and cry1F gene), using the leaves, while the remaining seven plots (12%) were positive for transgenic sequences in the seeds. The results indicated a significant level of overall transgene existence, which is consistent with gene flow from transgenic events. All of the field types (conventional maize, buffer zones, refuge, and Colombian landraces) showed evidence of a transgene presence. There are many problems that could increase the gene flow potential in Valle de San Juan, such as little respect for regulations (Colombian Decree 4525 on transgenic crops and biosafety), distance between transgenic and non-transgenic maize or use of refuge and/or buffer zones, high seed reuse and exchange and low technical assistance. Every policy decision must be made in light of scientific standards of judgment

Bacillus thuringiensis: en el manejo del agente de la pudrición blanda de la papa Erwinia carotovora

In Colombia the potato crop is the fourth in importance in the economy of the country, its production reached 300 million tons. Erwinia carotovora is a Gram-negative bacterium, facultative anaerobic which causes the soft rotting of the potato; it can potentially generate up to 100% damage in the crop, which causes large economic losses. It has been established that the bacterium Bacillus thuringiensis is able to suppress the virulence of E. caratovora because it produces N-acyl-homoserine-lactonasa, a powerful enzyme that degrades of N-acyl-homoserinolactonas, which are indispensable in the quorum-sensing mechanism of E. caratovora. This can be an important alternative for the control of the disease of the soft rotting of the potato. Considering the above, this article describes the process used by the bacterium B. thuringiensis to inhibit the activity of E. caratovora.En Colombia el cultivo de papa es el cuarto en importancia en la economía del país, y su producción alcanza las 300 millones de toneladas aproximadamente. Erwinia carotovora es una bacteria Gram negativa, anaeróbica facultativa causante de la pudrición blanda de la papa, puede llegar a generar hasta el 100% de daño en la cosecha, lo cual ocasiona grandes pérdidas económicas. Se ha establecido que la bacteria Bacillus thuringiensis es capaz de suprimir la virulencia de E. caratovora debido a que produce N-acil-homoserina-lactonasa, una potente enzima que degrada de N-acilhomoserinolactonas, que son indispensables en el mecanismo de quorum-sensing de E. caratovora. Esta circunstancia, puede ser una alternativa importante para el control de la enfermedad de la pudrición blanda de la papa. Considerando lo anterior, en este artículo se describe el proceso que emplea la bacteria Bacillus thuringiensis para inhibir la actividad de E. caratovora