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Tuberculosis (TB) remains an impactful infectious disease, leading to millions of deaths every year. Mycobacterium tuberculosis causes the formation of granulomas, which will determine, through the host-pathogen relationship, if the infection will remain latent or evolve into active disease. Early TB diagnosis is life-saving, especially among immunocompromised individuals, and leads to proper treatment, preventing transmission. This review addresses different approaches to diagnosing TB, from traditional methods such as sputum smear microscopy to more advanced molecular techniques. Integrating these techniques, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), has significantly improved the sensitivity and specificity of M. tuberculosis identification. Additionally, exploring novel biomarkers and applying artificial intelligence in radiological imaging contribute to more accurate and rapid diagnosis. Furthermore, we discuss the challenges of existing diagnostic methods, including limitations in resource-limited settings and the emergence of drug-resistant strains. While the primary focus of this review is on TB diagnosis, we also briefly explore the challenges and strategies for diagnosing non-tuberculous mycobacteria (NTM). In conclusion, this review provides an overview of the current landscape of TB diagnostics, emphasizing the need for ongoing research and innovation. As the field evolves, it is crucial to ensure that these advancements are accessible and applicable in diverse healthcare settings to effectively combat tuberculosis worldwide.
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Microorganisms play a vital role in biological wastewater treatment by converting organic and toxic materials into harmless substances. Understanding microbial communities' structure, taxonomy, phylogeny, and metabolic activities is essential to improve these processes. Molecular microbial ecology employs molecular techniques to study community profiles and phylogenetic information since culture-dependent approaches have limitations in providing a comprehensive understanding of microbial diversity in a system. Genomic advancements such as DNA hybridization, microarray analysis, sequencing, and reverse sample genome probing have enabled the detailed characterization of microbial communities in wastewater treatment facilities. This mini-review summarizes the current state of knowledge on the diversity of microorganisms in wastewater treatment plants, emphasizing critical microbial processes such as nitrogen and phosphorus removal.
Assuntos
Microbiota , Águas Residuárias , Filogenia , Genômica , Nitrogênio/metabolismo , Fósforo/metabolismo , Reatores Biológicos/microbiologia , Esgotos/microbiologiaRESUMO
Zika virus (ZIKV) is a teratogen that causes congenital anomalies, being linked to microcephaly in children exposed during pregnancy. Animal studies have been conducted to investigate the molecular mechanisms related to ZIKV teratogenesis. Although animal models can mimic the effects of ZIKV in human embryo development, few in vivo studies have addressed molecular changes following ZIKV infection in embryos. Moreover, few literature reviews have been conducted with these studies. The aim of this systematic review is to evaluate the molecular mechanisms of ZIKV teratogenesis determined from studies in animal models. PubMed/MEDLINE, EMBASE, Web of Science, and Scopus as well as grey literature were searched for studies that evaluated molecular alterations related to ZIKV teratogenesis which occurred during embryonic development. Nine studies were included: six with mice, one with mice and guinea pigs, one with pigs and one with chickens. In general, studies presented an unclear or high risk of bias for methodological criteria. Most of studies reported embryos exposed to ZIKV presenting microcephaly, reduced cortex thickness, and growth restriction. Different techniques were used to evaluated molecular changes in the animals following ZIKV infection: RNA sequencing, RT-qPCR, and in situ hybridization. It was found that common pathways are changed in most studies, being pathways related to immune response upregulated and those involved to neurodevelopment downregulated.
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Microcefalia , Malformações do Sistema Nervoso , Teratogênese , Infecção por Zika virus , Zika virus , Gravidez , Humanos , Criança , Feminino , Animais , Camundongos , Cobaias , Zika virus/fisiologia , Infecção por Zika virus/complicações , Galinhas , Modelos AnimaisRESUMO
Dogs are the most important reservoir of Leishmania infantum, the causal agent of visceral leishmaniasis in Brazil. Although lymphoid tissue is the most important biological tissue where amastigotes can be found, this paper describes the presence of L. infantum DNA in the milk of a lactating naturally infected female dog. This finding suggests the need for further studies to elucidate whether breastfeeding can be a route of infection.
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Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Cães , Feminino , Animais , Leishmania infantum/genética , Leite , Brasil , Lactação , Leishmaniose Visceral/veterináriaRESUMO
Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and ß-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and ß-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.
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Lyssavirus , Vírus da Raiva , Raiva , Doenças dos Roedores , Actinas , Animais , Lyssavirus/genética , Mamíferos , Camundongos , Raiva/diagnóstico , Raiva/epidemiologia , Raiva/veterinária , Vírus da Raiva/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Hereditary cancer predisposition syndromes are caused by germline pathogenic or likely pathogenic variants in cancer predisposition genes (CPG). The majority of pathogenic variants in CPGs are point mutations, but large gene rearrangements (LGRs) are present in several CPGs. LGRs can be much more difficult to characterize and perhaps they may have been neglected in molecular diagnoses. AREAS COVERED: We aimed to evaluate the frequencies of germline LGRs in studies conducted in different populations worldwide through a qualitative systematic review based on an online literature research in PubMed. Two reviewers independently extracted data from published studies between 2009 and 2020. In total, 126 studies from 37 countries and 5 continents were included in the analysis. The number of studies in different continents ranged from 3 to 48 and for several countries there was an absolute lack of information. Asia and Europe represented most of the studies, and LGR frequencies varied from 3.04 to 15.06% in different continents. MLPA was one of the methods of choice in most studies (93%). EXPERT OPINION: The LGR frequencies found in this review reinforce the need for comprehensive molecular testing regardless of the population of origin and should be considered by genetic counseling providers.
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Neoplasias da Mama , Neoplasias , Neoplasias da Mama/genética , Feminino , Rearranjo Gênico , Genes BRCA2 , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Mutação em Linhagem Germinativa , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , SíndromeRESUMO
The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.
O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.
Assuntos
Animais , Bovinos , Salmonella/isolamento & purificação , Búfalos , Leite/microbiologia , Fraude/prevenção & controle , Listeria monocytogenes/isolamento & purificação , Inocuidade dos Alimentos/métodos , Reação em Cadeia da Polimerase Multiplex/veterináriaRESUMO
Cancer genomics has evolved over the years from understanding the pathogenesis of cancer to screening the future possibilities of cancer occurrence. Understanding the genetic profile of tumors holds a prognostic as well as a predictive value in this era of therapeutic surveillance, molecular remission, and precision medicine. Identifying molecular markers in tumors is the current standard of approach, and requires an efficient combination of an accessible sample type and a profoundly sensitive technique. Liquid biopsy or cell-free DNA has evolved as a novel sample type with promising results in recent years. Although cell-free DNA has significant role in various cancer types, this review focuses on its application in Non-Hodgkin's Lymphoma. Beginning with the current concept and clinical relevance of minimal residual disease in Non-Hodgkin's lymphoma, we discuss the literature on circulating DNA and its evolving application in the realm of cutting-edge technology.
Assuntos
DNA Tumoral Circulante/sangue , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , Citometria de Fluxo , Marcadores Genéticos , Técnicas de Genotipagem , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Linfoma/sangue , Linfoma/genética , Linfoma não Hodgkin/sangue , Linfoma não Hodgkin/diagnóstico por imagem , Mutação , Neoplasia Residual/sangue , Neoplasia Residual/genética , Neoplasia Residual/patologia , Reação em Cadeia da Polimerase/métodos , Tomografia por Emissão de Pósitrons , Medicina de Precisão , Prognóstico , Transcriptoma , Translocação GenéticaRESUMO
Abstract Toxoplasmosis occurs worldwide causing economic losses to the animal production and problems to the public health. The study aimed to detect Toxoplasma gondii and Sarcocystis spp.in 141 meat products from commercial meat cuts of pork, beef, and kibbeh sold in commercial markets from Botucatu, SP, Brazil. Samples were bioassayed in mice to isolate the parasite, and the parasite DNA detected by PCR targeting the 529 base pairs repeat element region (PCR-529-bp). All samples resulted negative on bioassay, whereas PCR positive for 9 (6,38%), distributed as 5/48 beef, 3/49 pork, and 1/44 kibbeh. PCR-positive were investigated for the the parasite genotype using multiplex-, nested-, and RFLP-PCR for 11 markers (SAG1, 5'-3'SAG2, alt.SAG2, SAG3, B-TUB, GRA6, L358, c22-8, c29-6, PK1, Apico). Complete genotype was determined on just one PCR-positive sample that matched MAS, TgCkBr89 and TgCkBr147 isolates already identified. In addition, nested- and RFLP-PCR targeting 18S rRNA was run for all PCR-positive samples and, the products, sequenced and aligned to the GenBank at NCBI website. Four samples showed 100% homology with T. gondii (GenBank #L37415.1), three with Sarcocystis hominis (GenBank #AF006471.1), two Sarcocystis cruzi (GenBank #AF176934.1), and one Sarcocystis hirsuta (GenBank #AF006469.1), indicating the circulation of T. gondii and Sarcocystis spp.
Resumo A toxoplasmose está mundialmente distribuída e causa perdas na produção animal e problemas de saúde pública. Objetivou-se detectar Toxoplasma gondii e Sarcocystis spp. em 141 produtos cárneos de origem suína (49), bovina (48) e de quibe cru (44), comercializados em mercados de Botucatu, SP, Brazil. Realizou-se bioensaio das amostras em camundongos para isolamento do parasita, e detecção do DNA pela reação em cadeia pela polimerase, tendo como alvo a região do elemento repetitivo de 529 pares de bases (PCR-529-bp). Todas as amostras foram negativas ao bioensaio e 9 (6,38%) positivas à PCR, sendo 5/48 bovinas, 3/49 suínas e 1/44 quibe. Determinou-se a genotipagem das amostras positivas pela multiplex-, nested- e RFLP-PCR com 11 marcadores (SAG1, 5'-3'SAG2, alt.SAG2, SAG3, B-TUB, GRA6, L358, c22-8, c29-6, PK1, Apico). Obteve-se genótipo completo em uma amostra, semelhante a outros já identificados (MAS, TgCkBr89 e TgCkBr147). Nested- e RFLP-PCR do gene 18S rRNA das amostras positivas à PCR foram realizadas, e os produtos da nested-PCR, sequenciados e alinhados com dados do GenBank no NCBI. Quatro apresentaram 100% de homologia com T. gondii (L37415.1), duas Sarcocystis hominis (AF006471.1), duas Sarcocystis cruzi (AF176934.1), uma Sarcocystis hirsuta (AF006469.1), indicando a circulação de T. gondii e Sarcocystis spp.
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Animais , Ratos , Doenças dos Roedores , Toxoplasma/genética , Toxoplasmose Animal , Sarcocystis/genética , Brasil , DNA de Protozoário/genética , Genótipo , CarneRESUMO
After application, herbicides often reach the soil and affect non-target soil microorganisms, decreasing their population, diversity or affecting metabolic activity. Therefore, laboratory studies were performed to evaluate the effects of diuron, hexazinone and sulfometuron-methyl alone and mixed upon carbon transformation by soil microorganisms in clayey and sandy soils and the effect on bacterial diversity and structure. Control treatment without herbicide application was also performed. Sub-samples from the control and herbicide treatments (10 g - in triplicate) were collected before herbicide application and 7, 14, 28 and 42 days after treatment (DAT), then 1 mL of 14C-glucose solution was applied. The released 14CO2 was trapped in 2 M NaOH solution and the radioactivity was analyzed by liquid scintillation counting (LSC), 12 h after glucose application. The effect of herbicides on bacterial diversity was evaluated by T-RFLP. The experiment was conducted in a complete randomized design. Hexazinone did not affect 14CO2 evolution. Diuron showed a greater 14CO2 evolution in sandy and clayey soil, while sulfometuron-methyl led to an increase in sandy soil, at 42 DAT. A greater evolution of carbon was observed in the treatment with herbicide mixture in sandy soil, compared with the same treatment in clayey soil or control. However, the herbicide mixture application did not affect the soil biological activity measured by the respiration rate induced by substrate. On the other hand, the herbicide mixtures affected the bacterial diversity in both soils, being the strongest effect to diuron and sulfometuron-methyl in clayey soil and hexazinone in sandy soil.
Assuntos
Bactérias/efeitos dos fármacos , Diurona/toxicidade , Microbiologia do Solo , Compostos de Sulfonilureia/toxicidade , Triazinas/toxicidade , Bactérias/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Herbicidas/toxicidade , Polimorfismo de Fragmento de Restrição , Solo/química , Poluentes do Solo/toxicidadeRESUMO
La biología molecular (BM) es una ciencia que ha revolucionado el desarrollo científico en los últimos años. En el Instituto de Hematología e Inmunología (IHI) también ha evolucionado progresivamente conforme al avance tecnológico y adecuándose cada vez más al contexto científico internacional. Su historia se remonta al año 1966, con la creación del IHI y posteriormente del laboratorio de BM. En el año 2012, el departamento de BM y el laboratorio de citogenética, pasaron a formar parte de lo que hoy es el Centro de Tecnologías de Avanzada, un área con tecnología de punta que ha permitido actualizar la mayoría de las técnicas moleculares que se empleaban previamente, lo que garantiza mayor rapidez y confiabilidad de los resultados. Se introdujeron y perfeccionaron técnicas como la extracción y cuantificación de ácidos nucleicos, la electroforesis capilar y el FISH (del inglés: Fluorescence In Situ Hybridization) y se adquirieron modernas máquinas termocicladoras para la reacción en cadena de la polimerasa (PCR , siglas en inglés), materiales y reactivos. Con este esfuerzo mancomunado en estos 50 años, se han podido beneficiar hasta la fecha: 5 460 pacientes, estudiados en el laboratorio de BM, donde se determinan actualmente 10 marcadores moleculares, 12 estudios de FISH; además del cariotipo convencional y los estudios de quimerismo. Se ha alcanzado una media anual de 317 pacientes estudiados, en los últimos 5 años. Se cuenta con profesionales de alta calificación, lo que ha posibilitado liderar y colaborar en proyectos de investigación nacionales e internacionales, publicar innumerables artículos científicos, obtener premios relevantes y formar a los residentes de la especialidad de Hematología. Las perspectivas comprenden la incorporación de la PCR en tiempo real y la secuenciación para completar un nivel de diagnóstico a la altura de cualquier prestigioso centro internacional y así poder ofrecer un servicio de calidad a los pacientes(AU)
Molecular biology (MB) is a science that has revolutionized scientific development in recent years. It has also increasing progressively at the Institute of Hematology and Immunology (IHI) as adapting to technological advances and international scientific context. Its history dates back to 1966, with the IHI creation and subsequently the MB laboratory. Since then they have been many achievements in the field of diagnosis and research in Hematology. In 2012, the MB department with the cytogenetic laboratory was part of the Center for Advanced Technologies; an area with modern technology that has allowed change old studies by updated molecular techniques, ensuring greater speed and reliability of results. Techniques such as extraction and quantification of nucleic acids (NA), capillary electrophoresis and the FISH (Fluorescence in Situ Hybridization) were introduced, and modern thermocyclers for polymerase chain reaction (PCR), materials and reagents were acquired too. In these 50 years, 5 460 patients have been benefited to date. We study about 10 molecular markers, 12 FISH study, in addition to conventional karyotyping and chimerism studies in the MB lab at this moment. It has gone an annual average of 317 patients in the last 5 years. We have highly qualified professionals, which has made possible to lead and collaborate on national and international research, publishing numerous scientific articles, obtain relevant prizes of science and technology forum and directly contribute to the residents' formation in Hematology. Our future perspectives include the new technologies incorporation such as real-time PCR and sequencing, to complete a similar diagnostic level to any prestigious international center so we can provide quality service to our patients(AU)
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Humanos , Masculino , Feminino , Análise Citogenética/métodos , Hematologia/métodos , Biologia Molecular/história , Biologia Molecular/métodos , CubaRESUMO
Canine visceral leishmaniasis (CVL) is a worldwide anthropozoonosis caused by an intracellular parasite protozoan, Leishmania spp. In Americas, Leishmania (Leishmania) infantum is the causative agent, transmitted by sandflies, Lutzomyia longipalpis, after blood meal in domestic dogs, the main reservoir. The present study was aimed to determine the occurrence of Leishmania spp. and L. infantum in peripheral blood, and popliteal lymph node and bone marrow aspirate samples of 164 Brazilian stray dogs from an endemic area for CVL using the Polymerase Chain Reaction (PCR). For Leishmania spp., 56 (34.15%; 27.32-41.71%) blood, 102 (62.20%; 54.56-69.26%) lymph node, and 115 (70.12%; 62.71-76.60%) bone marrow samples tested positive, whereas 46 (28.05%; 21.74-35.38%), 94 (57.32%; 49.65-64.64%), and 114 (69.51%; 62.07-76.04%), respectively, resulted positive for L. infantum. Restriction Fragment Length Polymorphism (ITS1-RFLP) and sequencing were used to characterize the positive samples to Leishmania spp., but negative to L. infantum. Twenty (10 blood, 9 lymph node and 1 bone marrow) samples were characterized and matched the L. donovani complex species, with 99-100% similarity to L. donovani complex species (GenBank accession n.KC998879.1, JQ730002.1, GU045591.1, HQ830353.1, HM130608.1). The present study reports a high prevalence of stray dogs infected with leishmania species responsible for VL in the studied area, in which the observed diversity of leishmania species may contributes for further epidemiological studies.
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La expresión génica es el estudio de cómo el genotipo da lugar al fenotipo mediante la investigación de la cantidad de RNAm transcrito en un sistema biológico. Una gran cantidad de métodos fueron estandarizados para identificar variaciones en la expresión génica, incluyendo la hibridación sustractiva, differential display, análisis en serie de la expresión génica, la hibridación de microarrays, y la secuenciación por RNA-seq. La mayoría de las técnicas se han centrado en la investigación y diagnóstico del cáncer, produciendo una gran cantidad de datos, lo que permitió a entender la progresión del cáncer y las vías, descubrir y evaluar nuevas intervenciones de tratamiento, nuevas herramientas moleculares para el diagnóstico y el pronóstico, y analizar el tiempo de sobrevivencia en pacientes humanos y animales. De esta manera, las técnicas de expresión génica trajeron nuevas perspectivas importantes para el campo de la medicina veterinaria, y nuevas investigaciones centradas en oncología proporcionarán mucho más conocimiento acerca de las vías y la interacción de las células sanas y tumorales, mejorando las intervenciones diarias por los oncólogos y los clínicos.(AU)
Gene expression is the study of how the genotype gives rise to the phenotype by investigating the amount of transcribed mRNA in a biological system. A lot of methods have been standardized to identify the variation in gene expression, including subtractive hybridization, differential display, serial analysis of gene expression, microarray hybridization, and RNAseq sequencing. Most of techniques have been focused in cancer research and diagnosis, producing a huge amount of data, which allowed to understand the cancer progression and pathways, discover and evaluate new treatment interventions, new molecular tools for diagnosis and prognosis, and analyze the survival time in human and animal patients. In this way, gene expression techniques brought new important perspectives for the medical and veterinary fields, and further researches focusing oncology will provide much more knowledge concerning the pathways and interaction of healthy and tumor cells, improving the perspectives of the daily interventions by the oncologists and clinicians.(AU)
A expressão genética é o estudo de como o genótipo dá origem ao fenótipo a partir da investigação da quantidade de RNAm transcrito em um sistema biológico. Vários métodos já foram padronizados para identificar variações na expressão gênica, dentre eles a hibridização subtrativa, differential display, análise em série da expressão genética, hibridização de microarranjo, e sequenciamento por RNA-seq. A maioria das técnicas tem focado na pesquisa e diagnóstico do câncer, gerando enorme quantidade de dados, o que permitiu compreender a progressão do câncer e suas vias, descobrir e analisar novas intervenções terapêuticas, novas ferramentas moleculares para o diagnóstico e prognóstico, e analisar o tempo de sobrevivência em pacientes humanos e animais. Desta forma, as diferentes técnicas de expressão gênica trouxeram novas e importantes perspectivas para a área médica e veterinária, e novas pesquisas focadas em oncologia fornecerão muito mais conhecimento sobre as vias e interações entre células saudáveis e tumorais, melhorando as perspectivas das intervenções diárias pelos oncologistas e clínicos.(AU)
Assuntos
Humanos , Animais , Métodos , RNA Mensageiro/análise , Neoplasias/genética , Técnicas de Hibridização Subtrativa/veterináriaRESUMO
La expresión génica es el estudio de cómo el genotipo da lugar al fenotipo mediante la investigación de la cantidad de RNAm transcrito en un sistema biológico. Una gran cantidad de métodos fueron estandarizados para identificar variaciones en la expresión génica, incluyendo la hibridación sustractiva, differential display, análisis en serie de la expresión génica, la hibridación de microarrays, y la secuenciación por RNA-seq. La mayoría de las técnicas se han centrado en la investigación y diagnóstico del cáncer, produciendo una gran cantidad de datos, lo que permitió a entender la progresión del cáncer y las vías, descubrir y evaluar nuevas intervenciones de tratamiento, nuevas herramientas moleculares para el diagnóstico y el pronóstico, y analizar el tiempo de sobrevivencia en pacientes humanos y animales. De esta manera, las técnicas de expresión génica trajeron nuevas perspectivas importantes para el campo de la medicina veterinaria, y nuevas investigaciones centradas en oncología proporcionarán mucho más conocimiento acerca de las vías y la interacción de las células sanas y tumorales, mejorando las intervenciones diarias por los oncólogos y los clínicos.
Gene expression is the study of how the genotype gives rise to the phenotype by investigating the amount of transcribed mRNA in a biological system. A lot of methods have been standardized to identify the variation in gene expression, including subtractive hybridization, differential display, serial analysis of gene expression, microarray hybridization, and RNAseq sequencing. Most of techniques have been focused in cancer research and diagnosis, producing a huge amount of data, which allowed to understand the cancer progression and pathways, discover and evaluate new treatment interventions, new molecular tools for diagnosis and prognosis, and analyze the survival time in human and animal patients. In this way, gene expression techniques brought new important perspectives for the medical and veterinary fields, and further researches focusing oncology will provide much more knowledge concerning the pathways and interaction of healthy and tumor cells, improving the perspectives of the daily interventions by the oncologists and clinicians.
A expressão genética é o estudo de como o genótipo dá origem ao fenótipo a partir da investigação da quantidade de RNAm transcrito em um sistema biológico. Vários métodos já foram padronizados para identificar variações na expressão gênica, dentre eles a hibridização subtrativa, differential display, análise em série da expressão genética, hibridização de microarranjo, e sequenciamento por RNA-seq. A maioria das técnicas tem focado na pesquisa e diagnóstico do câncer, gerando enorme quantidade de dados, o que permitiu compreender a progressão do câncer e suas vias, descobrir e analisar novas intervenções terapêuticas, novas ferramentas moleculares para o diagnóstico e prognóstico, e analisar o tempo de sobrevivência em pacientes humanos e animais. Desta forma, as diferentes técnicas de expressão gênica trouxeram novas e importantes perspectivas para a área médica e veterinária, e novas pesquisas focadas em oncologia fornecerão muito mais conhecimento sobre as vias e interações entre células saudáveis e tumorais, melhorando as perspectivas das intervenções diárias pelos oncologistas e clínicos.
Assuntos
Humanos , Animais , Métodos , Neoplasias/genética , RNA Mensageiro/análise , Técnicas de Hibridização Subtrativa/veterináriaRESUMO
Abstract INTRODUCTION: Road-killed wild animals host zoonotic pathogens such as Toxoplasma gondii, offering a new opportunity for the epidemiological study of these infectious organisms. METHODS This investigation aimed to determine the presence of T. gondii and other apicomplexan parasites in tissue samples of 64 road-killed wild animals, using polymerase chain reaction (PCR). Positive samples were then typed by PCR-restriction fragment length polymorphism (RFLP) using 7 markers: SAG1, 5′-3′SAG2, SAG3, BTUB, c29-6, PK1, and Apico. PCR-RFLP targeting 18S ribosomal RNA (rRNA) genes was also performed on all samples to detect other apicomplexan parasites. RESULTS T. gondii DNA was detected in 16 tissue samples from 8 individual animals, as follows: 1 Cerdocyon thous (crab-eating fox), 1 Didelphis albiventris (white-eared opossum), 1 Lutreolina crassicaudata (lutrine opossum), 2 Myrmecophaga tridactyla (giant anteater), 1 Procyon cancrivorus (crab-eating raccoon), and 2 Sphiggurus spinosus (Paraguay hairy dwarf porcupine). Seven different T. gondii genotypes were identified, 6 of which were novel. Typing by 18S rRNA verified these 16 T. gondii-infected samples, and identified 1 Sarcocystis spp.-infected animal [Dasypus novemcinctus (nine-banded armadillo)]. The amplified T. gondii (GenBank accession No. L37415.1) and Sarcocystis spp. 18S rRNA products were confirmed by sequencing. CONCLUSIONS Our results indicate that T. gondii is commonly present in wild mammals, which act as sources of infection for humans and animals, including other wild species. The approach employed herein proved useful for detecting T. gondii and Sarcocystis spp. in the environment and identifying their natural reservoirs, contributing to our understanding of host-parasite interactions.
Assuntos
Animais , Toxoplasma/genética , DNA de Protozoário/genética , Sarcocystis/genética , Animais Selvagens/parasitologia , Mamíferos/parasitologia , Toxoplasma/isolamento & purificação , Brasil , Reação em Cadeia da Polimerase , Sarcocystis/isolamento & purificação , GenótipoRESUMO
Toxoplasma gondii has a worldwide distribution with different genotypes reported in animals and humans. The parasite is of great importance to food production and public health, highlighted by the high diversity of hosts, i.e. ostriches. This study aimed to determine the prevalence of T. gondii infection in ostriches from a Brazilian slaughterhouse, the genotype, and the associated risk factors. T. gondii antibodies were detected in 38/344 (11.05%) serum samples using the modified agglutination test using formalin-fixed tachyzoites (MAT-HS); the parasite was isolated from 14/38 (36.84%) ostrich brain samples using the mouse bioassay; and the DNA was detected from 25/38 (65.79%), using PCR. In farms, the water tank was considered the main risk factor (OR=141.87; p-value<0.05), and oocysts were detected in 30% (6/20) in soil of paddocks before animals were slaughtered (1st sampling), and 40% (8/20) one-year after (2nd sampling) using microscopy and PCR. Non-ostrich fecal samples on the ground resulted negative. Bioassay isolation was confirmed by PCR. All PCR positive samples were sequenced and resulted in 100% homology to Toxoplasma gondii repetitive DNA sequence (GenBank access number EF648168-1). These samples were also typed through RFLP-PCR using 11 markers: SAG1, SAG2 (5'-3'SAG2 and alt.SAG2), SAG3, BTUB, GRA6, L358, c22-8, c29-6, PK1, Apico and CS3. Two isolates had a complete genotype, typed from the ostrich tissue. In ostrich samples, the parasite load ranged from 19,043 (TgOsBr1, avirulent) to 54,829 parasitesmL(-1) (TgOsBr2, virulent) using qPCR, whereas soil samples ranged from 11 to 2,275 parasitesmL(-1). Both typed isolates resulted on atypical clones, one previously reported to cause congenital toxoplasmosis in Brazilian patients (TgOsBr1, ToxoDB #206). Thus, these findings support the occurrence of T. gondii in slaughtered ostriches from Brazil, ostriches as sentinel for environmental contamination with T. gondii, the genotypic variability in Brazilian isolates, and the first isolation and genotyping of T. gondii from Brazilian slaughtered ostriches.
Assuntos
Doenças das Aves/epidemiologia , Doenças das Aves/parasitologia , Struthioniformes/parasitologia , Toxoplasma/genética , Toxoplasmose Animal/epidemiologia , Toxoplasmose Animal/parasitologia , Matadouros , Animais , Anticorpos Antiprotozoários/sangue , Brasil/epidemiologia , DNA de Protozoário/genética , Variação Genética , Genótipo , Camundongos , Tipagem Molecular , Oocistos , Carga Parasitária , Prevalência , Fatores de Risco , Homologia de Sequência do Ácido NucleicoRESUMO
Canine Heartworm Disease (CHD) is a mosquito-borne disease caused by Dirofilaria immitis. In this study, two mature adult-senior dogs from a non-endemic area to CHD presented clinical signs suggestive to the disease. The first one presented skin lesions, loss of appetite, weakness, pale mucosa membrane, and hyperthermia, whereas the second one presented severe ascites, anorexia and exercise intolerance, lateral decumbency, and marked heart murmurs. Both presented tachypnea, thrombocytopenia, leukocytosis, and microfilaremia. Multiplex-PCR (COI gene) resulted positive to D. immitis research in both cases, confirmed by sequencing, with 98% homology to D. immitis (Gen Bank accession n.AJ537512-1). In addition, both animals have never had any prophylactic treatment to CHD, and no reports about traveling to coastal areas. This study reported two unusual cases of D. immitis infection in non-endemic area from Brazil.
A dirofilariose canina (CHD) é uma doença transmitida por mosquitos e causada por Dirofilaria immitis. No presente estudo, dois cães de idade adulta a idoso de área não endêmica apresentaram sinais clínicos sugestivos da doença. O primeiro apresentou lesões de pele, perda de apetite, fraqueza, mucosas pálidas e hipertermia, enquanto o segundo apresentou severo quadro de ascite, anorexia e intolerância ao exercício, decúbito lateral, e murmúrios cardíacos acentuados. Ambos apresentaram taquipneia, trombocitopenia, leucocitose e microfilaremia. A pesquisa por D. immitis pela multiplex-PCR (COI gene) resultou positiva em ambos os casos, confirmada pelo sequenciamento, com 98% de homologia com D. immitis (Gen Bank n. AJ537512-1). Nenhum dos animais havia sido submetido a tratamento profilático para CHD e não havia relatos de viagens para regiões litorâneas. Assim, o presente estudo reporta dois casos raros de infecção por D. immitis em área brasileira não endêmica para a doença.
La dirofilariosis canina (CHD) es una enfermedad transmitida por mosquitos y causada por Dirofilaria immitis. En este estudio, dos perros de edad adulta a anciano, de área no endémica presentaron signos clínicos de la enfermedad. El primero presentó lesiones en la piel, pérdida del apetito, debilidad, palidez de mucosas e hipertermia, mientras el otro presentó severa ascitis, anorexia e intolerancia al ejercicio, decúbito lateral, y soplos cardíacos acentuados. Ambos presentaron taquipnea, trombocitopenia, leucocitosis y microfilaremia. La investigación de D. immitis por multiplex-PCR (gen COI) resultó positivo en ambos casos, confirmados por la técnica de secuenciación, con 98% de homología con D. immitis (Gen Bank n.AJ537512-1). Ninguno de los animales había sido sometido al tratamiento profiláctico para CHD, y sin relatos de viajes a regiones costeras. El presente estudio reporta dos casos raros de la infección por D. immitis en zona no endémica de Brasil.
Assuntos
Animais , Cães , Dirofilariose/classificação , Dirofilariose/diagnóstico , Doenças Endêmicas/veterinária , Dirofilaria immitis , Cães/anormalidadesRESUMO
Canine Heartworm Disease (CHD) is a mosquito-borne disease caused by Dirofilaria immitis. In this study, two mature adult-senior dogs from a non-endemic area to CHD presented clinical signs suggestive to the disease. The first one presented skin lesions, loss of appetite, weakness, pale mucosa membrane, and hyperthermia, whereas the second one presented severe ascites, anorexia and exercise intolerance, lateral decumbency, and marked heart murmurs. Both presented tachypnea, thrombocytopenia, leukocytosis, and microfilaremia. Multiplex-PCR (COI gene) resulted positive to D. immitis research in both cases, confirmed by sequencing, with 98% homology to D. immitis (Gen Bank accession n.AJ537512-1). In addition, both animals have never had any prophylactic treatment to CHD, and no reports about traveling to coastal areas. This study reported two unusual cases of D. immitis infection in non-endemic area from Brazil.(AU)
A dirofilariose canina (CHD) é uma doença transmitida por mosquitos e causada por Dirofilaria immitis. No presente estudo, dois cães de idade adulta a idoso de área não endêmica apresentaram sinais clínicos sugestivos da doença. O primeiro apresentou lesões de pele, perda de apetite, fraqueza, mucosas pálidas e hipertermia, enquanto o segundo apresentou severo quadro de ascite, anorexia e intolerância ao exercício, decúbito lateral, e murmúrios cardíacos acentuados. Ambos apresentaram taquipneia, trombocitopenia, leucocitose e microfilaremia. A pesquisa por D. immitis pela multiplex-PCR (COI gene) resultou positiva em ambos os casos, confirmada pelo sequenciamento, com 98% de homologia com D. immitis (Gen Bank n. AJ537512-1). Nenhum dos animais havia sido submetido a tratamento profilático para CHD e não havia relatos de viagens para regiões litorâneas. Assim, o presente estudo reporta dois casos raros de infecção por D. immitis em área brasileira não endêmica para a doença.(AU)
La dirofilariosis canina (CHD) es una enfermedad transmitida por mosquitos y causada por Dirofilaria immitis. En este estudio, dos perros de edad adulta a anciano, de área no endémica presentaron signos clínicos de la enfermedad. El primero presentó lesiones en la piel, pérdida del apetito, debilidad, palidez de mucosas e hipertermia, mientras el otro presentó severa ascitis, anorexia e intolerancia al ejercicio, decúbito lateral, y soplos cardíacos acentuados. Ambos presentaron taquipnea, trombocitopenia, leucocitosis y microfilaremia. La investigación de D. immitis por multiplex-PCR (gen COI) resultó positivo en ambos casos, confirmados por la técnica de secuenciación, con 98% de homología con D. immitis (Gen Bank n.AJ537512-1). Ninguno de los animales había sido sometido al tratamiento profiláctico para CHD, y sin relatos de viajes a regiones costeras. El presente estudio reporta dos casos raros de la infección por D. immitis en zona no endémica de Brasil.(AU)
Assuntos
Animais , Dirofilariose/diagnóstico , Dirofilariose/classificação , Doenças Endêmicas/veterinária , Cães/anormalidades , Dirofilaria immitisRESUMO
This review presents the principal methods used in taxonomic studies of rumen ciliates: live observation, Lugol staining, fixation and staining with methyl-green formalin saline (MFS) solution, protargol staining, silver carbonate impregnation, scanning electron microscopy and molecular techniques. Mastering these techniques is essential for successful research on the taxonomy of rumen ciliates. No single technique reveals all of the characteristics required for a complete description of a rumen ciliate; therefore, it is necessary to combine the use of these techniques as appropriate to the rumen ciliate group under study. Tables are provided to summarize: 1) morphological methods more appropriate for revealing morphological structures of interest, 2) morphological methods indicated for each group of rumen ciliates, and 3) main primers used for PCR amplification of the 18S rDNA of rumen ciliates.
Assuntos
Cilióforos/classificação , Rúmen/parasitologia , Animais , Cilióforos/ultraestrutura , Classificação , Coloração e RotulagemRESUMO
The diversity of lactic acid bacteria (LAB) associated with chicha, a traditional maize-based fermented alcoholic beverage from Northwestern Argentina, was analyzed using culture-dependent and culture-independent approaches. Samples corresponding to 10 production steps were obtained from two local producers at Maimará (chicha M) and Tumbaya (chicha T). Whereas by culture-dependent approach a few number of species (Lactobacillus plantarum and Weissella viridescens in chicha M, and Enterococcus faecium and Leuconostoc mesenteroides in chicha T) were identified, a higher quantitative distribution of taxa was found in both beverages by pyrosequencing. The relative abundance of OTUs was higher in chicha M than in chicha T; six LAB genera were common for chicha M and T: Enterococcus, Lactococcus, Streptococcus, Weissella, Leuconostoc and Lactobacillus while Pediococcus only was detected in chicha M. Among the 46 identified LAB species, those of Lactobacillus were dominant in both chicha samples, exhibiting the highest diversity, whereas Enterococcus and Leuconostoc were recorded as the second dominant genera in chicha T and M, respectively. Identification at species level showed the predominance of Lb. plantarum, Lactobacillus rossiae, Leuconostoc lactis and W. viridescens in chicha M while Enterococcus hirae, E. faecium, Lc. mesenteroides and Weissella confusa predominated in chicha T samples. In parallel, when presumptive LAB isolates (chicha M: 146; chicha T: 246) recovered from the same samples were identified by ISR-PCR and RAPD-PCR profiles, species-specific PCR and 16S rRNA gene sequencing, most of them were assigned to the Leuconostoc genus (Lc. mesenteroides and Lc. lactis) in chicha M, Lactobacillus, Weissella and Enterococcus being also present. In contrast, chicha T exhibited the presence of Enterococcus and Leuconostoc, E. faecium being the most representative species. Massive sequencing approach was applied for the first time to study the diversity and evolution of microbial communities during chicha manufacture. Although differences in the LAB species profile between the two geographically different chicha productions were observed by culturing, a larger number for predominant LAB species as well as other minorities were revealed by pyrosequencing. The fine molecular inventory achieved by pyrosequencing provided more precise information on LAB population composition than culture-dependent analysis processes.