Estimation of genetic variation in closely related cycad species in Ceratozamia (Zamiaceae: Cycadales) using rapds markers / Variación genética en especies de cícadas estrechamente relacionadas en Ceratozamia (Zamiaceae: Cycadales) mediante el uso de RAPDs

AbstractThe Ceratozamianorstogii complex from Southern Mexico is made up of four closely related taxa and occurs in similar habitats (Quercus forest). All have linear-lanceolate leaflets with great similarity between them, especially in juvenile stages, but differentiate with age. There has been debate regarding delimitation of species due to character loss in herbarium specimens. The aim of this study was to determine the genetic variation, and to measure genetic similarity between the four taxa. We studied populations in Cintalapa (Chiapas) for C. alvarezii and C. norstogii; the Sierra Atravesada (Oaxaca) for C. chimalapensis, and Villa Flores (Chiapas) for C. mirandae. One population for each taxon was sampled (only one population is known for C. alvarezii) 11-15 randomly chosen adult individuals were sampled. Twenty-eight primers were tested of which five were polymorphic using the RAPD'S technique. The data were analyzed using Bayesian methods. Results revealed low genetic diversity, and a differentiation was found between species, suggesting a recent divergence. A previous morphological and anatomical study on the complex has found the taxa to be distinct. However, the results of this study have shown that the C. norstogii species complex is in a divergence process, probably through genetic drift and founder effects. Rev. Biol. Trop. 65 (1): 305-319. Epub 2017 March 01.

ResumenLos cuatro taxa que componen el complejo Ceratozamia norstogii de especies en el sur de México están estrechamente relacionados y se dan en hábitats similares (bosque de Quercus). Todos tienen folíolos linear-lanceolados con gran similitud entre ellos, sobre todo en las etapas juveniles, pero se diferencian con la edad. Ha habido un debate en relación con la delimitación de especies debido a la pérdida de caracteres en especímenes de herbario. Los objetivos de este estudio son determinar la variación genética y medir la similitud genética entre los cuatro taxones en el complejo. Las poblaciones estudiadas están en; Cintalapa, Chiapas para C. alvarezii y C. norstogii, la Sierra Atravesada, Oaxaca para C. chimalapensis y Villa Flores, Chiapas para C. mirandae. Se tomaron muestras de una población de cada taxón (sólo una población es conocida para C. alvarezii) 11-15 individuos adultos elegidos al azar fueron muestreados. Veintiocho primers fueron probados, de los cuales cinco fueron polimórficos mediante la técnica RAPD's. Los datos fueron analizados utilizando métodos bayesianos. Los resultados revelaron baja diversidad genética y la diferenciación encontrada entre las especies sugiere una divergencia reciente. Un estudio morfológico y anatómico anterior en el complejo encontró que los taxa son distintos. Sin embargo, los resultados del presente estudio han demostrado que el complejo C. norstogii aun se encuentra en un proceso de divergencia, probablemente a través de deriva genética y efectos de fundador.

Genetic characterization and authentication of Gardenia jasminoides in different regions of China by using improved RAPD analysis.

Gardenia jasminoides is a common garden medicinal plant known for its anticancer, anti-inflammatory, anti-thrombic, anti-fibrotic, antiviral, hepatoprotective, lung-protective, renal-protective, retina-protective and neuroprotective activities. It is found in several regions of the world, including China, but information about its genetic characteristics is limited. Here, we employed an improved method of random amplified polymorphic DNA (RAPD) analysis (with increased RAMP time) to investigate the genetic link between G. jasminoides samples collected from six different regions of Southern China. Total 26 RAPD primers were selected randomly, among which 23 primers generated reproducible polymorphic amplification bands. A total of 174 bands were obtained, where each primer had amplified 5-13 bands with an average of 7.56 bands per primer. The band size ranged approximately 150-2200 bp. Cluster dendrogram was obtained based on the improved RAPD amplification profiles, which showed that the similarity coefficients among six varieties of G. jasminoides ranged 0.67-0.88. To our knowledge, this is the first report of genetic characterization of G. jasminoides using improved RAPD analysis, which may be useful for the preservation of genetic diversity and identification of Gardenia population.

Using molecular markers to assess Streptococcus mutans variability and the biological risk for caries

AIM: To characterize the genetic variability of Streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. METHODS: S. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16S-23S intergenic spacer region. The genetic variability of the isolates was first assessed using Random Amplified Polymorphic DNA (RAPD) markers. Next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase B (gtfB). RESULTS: Characterization using RAPD markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. By sequencing the 16S-23S intergenic region, it was found that all of the isolates belonged to the species S. mutans. Based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfB gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. CONCLUSIONS: The partial sequencing of the gtfB gene can be a useful tool for elucidating the colonization patterns of S. mutans. As amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children...

Comparison of primers for RAPD-PCR from environmental isolates of Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii complex

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e.g., CAV2 for C. albidus) may not to be applied to some species.

Serotyping & molecular characterization for study of genetic diversity among seafood associated nontyphoidal Salmonella serovars.

Background & objectives: Infections due to seafood associated Salmonella serovars are great risk to public health. Different phenotypic characteristics have been used previously for epidemiological investigation of Salmonella. Beyond the phenotypic characterization, a reliable genetic level discriminatory method is required. Therefore, this study was attempted to use different phenotypic and molecular fingerprinting methods for investigation of genetic diversity among seafood associated nontyphoidal Salmonella serovars. Methods: Fifty eight seafood associated Salmonella isolates were included in this study. All isolates were serotyped and epidemiological investigation was carried out using molecular fingerprinting methods, random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR) along with whole cell protein profiling using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in our study. Results: Among the 58 Salmonella isolates, S. Weltevreden was observed to be the most predominant serovar. Typing of Salmonella serovars using RAPD and ERIC-PCR suggested the existence of a genetic diversity. Though both PCR based techniques were found to have a good discriminatory index, a better discriminatory ability was observed when the results obtained by the two techniques were combined and taken for composite analysis. Protein profiling of whole cells using SDS-PAGE demonstrated the presence of several bands with two bands of sizes 38 kDa and 46 kDa common among all 58 isolates. Interpretation & conclusions: Our study shows that use of protein profiling in combination with established typing methods such as RAPD and ERIC-PCR may provide useful information in typing of non-typhoidal Salmonella isolates associated with seafood and to develop strategies to protect public from Salmonella infections.

Cenário ecoepidemiológico da doença de Chagas no entorno do Parque Nacional da Serra do Cipó, Minas Gerais,Brasil

A doença de Chagas (DC) é uma enfermidade causada pelo protozoário Trypanosoma cruzi,cuja principal forma de transmissão é através das fezes de triatomíneos infectados. O Panstrongylus megistus atualmente é o principal vetor do Brasil, sendo responsável pela transmissão da DC na região da Serra do Cipó, MG, na década de 80. O P. megistus possui alta capacidade de reinfestação das casas, exigindo permanente vigilância contra a instalação de novos focos. O peridomicílio apresenta grande importância para a manutenção de triatomíneos e do T. cruzi circulando entre barbeiros e os animais que o frequentam. O objetivo do trabalho foi avaliar o cenário ecoepidemiológico da doença de Chagas noentorno do Parque Nacional da Serra do Cipó (ParnaCipó), visando subsidiar o programa de controle do P. megistus. O estudo se realizou nos municípios de Jaboticatubas e Santana do Riacho que, juntamente com Morro do Pilar e Itambé do Mato Dentro, integram o ParnaCipó. O perfil de infestação pelo P. megistus foi determinado através de captura de triatomíneos nas unidades domiciliares (setembro de 2007 a setembro de 2010) e capturas silvestres, que também incluiram avaliação da infecção em marsupiais e roedores.

Cães levados à campanha de vacinação antirrábica de Jaboticatubas foram examinados a fim de determinar sua importância epidemiológica na região. Exemplares de P. megistus capturados foram utilizados para análise populacional pela morfometria geométrica. Os exemplares provenientes de Jaboticatubas foram ainda analisados por RAPD para estudo populacional. As cepas isoladas dos reservatórios e vetores foram caracterizadas como pertencentes ao grupo T. cruzi I ou T. cruzi II utilizando‐se DNA satélite como alvo. A maioria dos exemplares P. megistus foi capturada em galinheiros; no intradomicílio o ecótopo preferencial foi o quarto. Foram realizadas pesquisas em 15 áreas silvestres com a captura de 105 mamíferos, com taxa de infecção de 6,7%; todas as cepas foram caracterizadas como T. cruzi I. A prevalência em cães foi de 2,4%. Em palmeiras foram capturados Rhodnius neglectus, P. megistus e Triatoma sordida. Dentre as cepas isoladas de triatomíneos domiciliados e silvestres, apenas uma cepa proveniente do ambiente domiciliar foi caracterizada como T. cruzi II, as demais, T. cruzi I. A morfometria diferenciou a população silvestre das domiciliares. A RAPD demonstrou grande variabilidade dos P. megistus de Jaboticatubas, entretanto, sem diferenciação populacional. Frente aos resultados, podemos concluir que a população rural dos dois municípios permanece sob o risco de infecção pelo T. cruzi, uma vez que são encontrados vetores e reservatórios infectados no ambiente artificial e natural, e podem infestar as habitações a partir de diversos focos, domiciliares ou silvestres.

Genomic instability in human actinic keratosis and squamous cell carcinoma

OBJECTIVE: To compare the repetitive DNA patterns of human actinic keratoses and squamous cell carcinomas to determine the genetic alterations that are associated with malignant transformation. INTRODUCTION: Cancer cells are prone to genomic instability, which is often due to DNA polymerase slippage during the replication of repetitive DNA and to mutations in the DNA repair genes. The progression of benign actinic keratoses to malignant squamous cell carcinomas has been proposed by several authors. MATERIAL AND METHODS: Eight actinic keratoses and 24 squamous cell carcinomas (SCC), which were pair-matched to adjacent skin tissues and/or leucocytes, were studied. The presence of microsatellite instability (MSI) and the loss of heterozygosity (LOH) in chromosomes 6 and 9 were investigated using nine PCR primer pairs. Random Amplified Polymorphic DNA patterns were also evaluated using eight primers. RESULTS: MSI was detected in two (D6S251, D9S50) of the eight actinic keratosis patients. Among the 8 patients who had squamous cell carcinoma-I and provided informative results, a single patient exhibited two LOH (D6S251, D9S287) and two instances of MSI (D9S180, D9S280). Two LOH and one example of MSI (D6S251) were detected in three out of the 10 patients with squamous cell carcinoma-II. Among the four patients with squamous cell carcinoma-III, one patient displayed three MSIs (D6S251, D6S252, and D9S180) and another patient exhibited an MSI (D9S280). The altered random amplified polymorphic DNA ranged from 70 percent actinic keratoses, 76 percent squamous cell carcinoma-I, and 90 percent squamous cell carcinoma-II, to 100 percent squamous cell carcinoma-III. DISCUSSION: The increased levels of alterations in the microsatellites, particularly in D6S251, and the random amplified polymorphic DNA fingerprints were statistically significant in squamous cell carcinomas, compared with actinic keratoses. CONCLUSION: The overall alterations that were observed in the repetitive DNA of actinic keratoses and squamous cell carcinomas indicate the presence of a spectrum of malignant progression.

Molecular techniques for the study and diagnosis of parasite infection

In parasitology, routine laboratory diagnosis involves conventional methods, such as optical microscopy, used for the morphological identification of parasites. Currently, molecular biology techniques are increasingly used to diagnose parasite structures in order to enhance the identification and characterization of parasites. The objective of the present study was to review the main current and new diagnostic techniques for confirmation of parasite infections, namely: polymerase chain reaction (PCR), real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), Luminex xMAP, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism (RFLP), in addition to microsatellites. Molecular assays have comprehensively assisted in the diagnosis, treatment and epidemiological studies of parasitic diseases that affect people worldwide, helping to control parasitic disease mortality.

Identification of the diagnostic and epidemiological potential of camel strain G6 in human cystic echinococcosis

Recent molecular studies detected the presence of camel G6 genotype in human samples in different countries including Egypt. However, none of them studied the diagnostic and epidemiological role of camel G6 genotype in patients' sera with cystic echinococcosis [CE]. Detection of camel G6 strain in patients' sera by polymerase chain reaction [PCR] and determination of changes in the genotype profile that might be influenced by the predominant transmission cycle during a certain time. Eighty subjects were divided into 2 main groups with 3 subgroups in each: Group I [31 CE cases with positive G6 PCR in parasite material obtained from their cysts] subdivided into Group IA [21 hepatic CE], Group IB [5 pulmonary CE], Group IC [5 multiple organ CE], Group II [49 control subjects] including Group IIA [29 patients with other parasitic diseases], Group IIB [10 patients with space occupying lesions] and Group IIC [10 healthy individuals]. DNA was extracted from CE patients' sera for amplification and sequencing. Hot-start specific G6 PCR for patients' sera [PCRs] revealed that all CE cases [100%] were of G6 genotype, with 100% sensitivity and 100% specificity and a specific band at 254 bp. Indirect hemagglutination test [IHAT] showed 61.29% sensitivity and 95.92% specificity. DNA sequencing of the amplified DNA fragments of patient's sera showed 100% homology with extracts from parasite materials taken from their own cysts [Gen Bank under the accession no. from GQ476732 to GQ476735], as well as with that of an Argentinean reference strain [provided from WHO reference laboratory]

Caracterização genética de Brycon orbignyanus utilizando o sistema seminatural / Genetic characterization of Brycon orbignyanus using the semi-natural system

Avaliou-se o efeito do sistema seminatural na diversidade genética de um estoque de Brycon orbignyanus, utilizado em programas de repovoamento, com o marcador molecular RAPD. Vinte e quatro reprodutores, 12 machos e 12 fêmeas e 95 larvas da progênie foram analisados. Os nove primers utilizados produziram 90 fragmentos, dos quais 94,4 por cento foram polimórficos. Houve diferença significativa na frequência de 20 dos 90 fragmentos entre os reprodutores e sua progênie sem a presença de fragmentos exclusivos. O índice de diversidade genética de Shannon, a porcentagem de fragmentos polimórficos e a diversidade genética de Nei foram mais altos nos indivíduos da progênie. A similaridade genética foi maior nos indivíduos do estoque de reprodutores. A análise de variância molecular mostrou que a maior parte da variação está dentro de cada grupo (89,1 por cento) e não entre os grupos (10,9 por cento). A identidade e a distância genética entre os estoques foram de 0,944 e 0,057, respectivamente. Assim, a utilização do sistema seminatural evitou a mortalidade de reprodutores B. orbignyanus e conservou a variabilidade genética da progênie.

The effect of the semi-natural system on the genetic diversity of a Brycon orbignyanus stock, used in stock enhancement programs, was evaluated with the RAPD molecular marker. Twenty-four broodstocks - 12 males and 12 females - and 95 larvae of the offspring were analyzed. The nine used primers produced 90 fragments, of which 94.4 percent were polymorphic. There was significant difference in the frequency of 20 out of the 90 fragments between the broodstocks and their offspring without the presence of exclusive fragments. The Shannon genetic diversity index, the percentage of polymorphic fragments and the Nei gene diversity were higher in the offspring individuals. Genetic similarity was higher in broodstock individuals. The analysis of molecular variance results showed that the major part of the genetic variation is within the groups (89.1 percent) and not between them (10.9 percent). The identity and genetic distance between the groups were 0.944 and 0.057, respectively. Like this, the use of the semi-natural system avoided the mortality of B. orbignyanus broodstocks and conserved the genetic variability of the offspring.

Monitoramento da variabilidade genética de pacu, Piaractus mesopotamicus, do programa de aumento de estoque do rio Paranapanema / Monitoring of the genetic variability of pacu, Piaractus mesopotamicus, of the stock enhancement program of the Paranapanema River

Avaliou-se a variabilidade genética dos estoques de reprodutores e dos peixes jovens de Piaractus mesopotamicus de três pisciculturas do estado do Paraná, utilizadas no programa de aumento de estoque de peixes no rio Paranapanema. Foi utilizado o marcador RAPD para avaliar as amostras do estoque de reprodutores e dos peixes jovens das pisciculturas de Palotina, Cambará e Andirá. A porcentagem de fragmentos polimórficos e o índice de diversidade genética de Shannon dos estoques de reprodutores variaram de 75,0 por cento a 71,4 por cento e de 0,434 a 0,376, respectivamente. Os peixes jovens das pisciculturas apresentaram valores mais elevados para ambos os parâmetros, com exceção da piscicultura de Palotina, na qual o índice de diversidade genética de Shannon foi semelhante. Os estoques de reprodutores apresentaram alta variabilidade genética, e esta foi mantida nos peixes jovens.

The genetic variability of broodstocks and juveniles of Piaractus mesopotamicus raised in three hatchery stations in Parana state, that were used in the fish stock enhancement program of the Paranapanema River, was estimated. The RAPD marker was used to evaluate samples taken from broodstocks and juveniles in the hatchery stations of Palotina, Cambará, and Andirá cities. The percentage of polymorphic fragments and the Shannon genetic diversity index of broodstocks ranged from 75.0 percent to 71.4 percent and from 0.434 to 0.376, respectively. The juveniles of the hatchery stations presented higher values for both parameters, except in the hatchery station of Palotina in which the Shannon genetic diversity index was similar. The broodstocks presented high genetic variability, and this was maintained in the juveniles.

Diversidade genética de estoques de reprodutores de Colossoma macropomum / Genetic diversity of Colossoma macropomum broodstocks

Analisou-se a diversidade genética de estoques de reprodutores de Tambaqui (Colossoma macropomum), mediante o uso de marcador RAPD, utilizando-se 10 primers para analisar 30 amostras do estoques de reprodutores das pisciculturas de Boa Esperança e Vale Verde, localizadas no Estado de Rondônia. A porcentagem de fragmentos polimórficos e o índice de diversidade genética de Shannon foram altos nos dois estoques de reprodutores. O estoque de reprodutores de Boa Esperança apresentou um fragmento exclusivo. A diferenciação genética foi baixa e o número de migrantes por geração foi alto entre os estoques de reprodutores. O dendrograma não separou os indivíduos dos estoques de reprodutores em grupos distintos. Há alta variabilidade genética nos estoques de reprodutores, um pouco inferior no estoque de Vale Verde, e há grande proximidade genética entre os indivíduos dos estoques de reprodutores.

The genetic diversity of tambaqui (Colossoma macropomum) broodstocks from two hatchery station in Rondônia State was studied by the RAPD marker. Ten primers were used to analyze 30 broodstocks samples from the hatchery stations of Boa Esperança and Vale Verde. The polymorphic fragments percentage and Shannon genetic diversity index were high in the two broodstocks. The Boa Esperança broodstock presented an exclusive fragment. The genetic differentiation was low and the number of migrants per generation was high among the broodstocks. The dendrogram did not separate the broodstocks individuals in different groups. The results indicate a high genetic variability in the broodstocks, being a little bit lower in the Vale Verde broodstock. Besides, there is a genetic proximity among the broodstocks.

Caracterização fenotípica e molecular de amostras de Burkholderia mallei isoladas na Região Nordeste do Brasil / Phenotypic and molecular characterization of Burkholderia mallei isolated in northeastern Brazil

Objetivou-se com este trabalho realizar o estudo bioquímico e molecular de amostras de Burkholderia mallei isoladas de eqüídeos com diagnóstico clínico e sorológico para o mormo e provenientes da Região Metropolitana do Recife-PE e Zona da Mata dos Estados de Alagoas e Pernambuco. Foram realizadas as técnicas microbiológicas para o isolamento e identificação fenotípica de B. mallei e as técnicas moleculares de ribotipagem-PCR e RAPD-PCR. Das oito amostras estudadas, quatro apresentaram pequenas variações fenotípicas. Nas técnicas moleculares, as amostras formaram quatro grupos de diferentes perfis ribotípicos, demonstrando também quatro perfis genotípicos. Houve associação nos resultados da Ribotipagem-PCR e RAPD-PCR. As variações nos perfis ribotípicos e genotípicos foram associadas às diferentes regiões estudadas. De acordo com os resultados obtidos, conclui-se que as pequenas variações bioquímicas não estão associadas aos diferentes perfis moleculares e que essas diferenças demonstram uma heterogeneidade que está associada à procedência das amostras, indicando que a infecção nos animais ocorre por clones diferentes das amostras analisadas.

The objective of this paper was to study the molecular performance and phenotypic characterization of Burkholderia mallei isolated from horses with clinical and serological diagnosis of glanders, originating from the Metropolitan District of Recife and Zona da Mata of Pernambuco and Alagoas. The isolation and biochemical identification of B. mallei was carried out by microbiological and molecular techniques of PCR-fingerprinting and RAPD-PCR. From the eight samples studied, four showed little phenotype variations. In the molecular tests, the samples formed 4 groups of different ribotype profiles and 4 genotype profiles. There was some association of PCR-fingerprinting with RAPD-PCR results. It was concluded that the slight biochemical variations were not associated with different molecular profiles. They also indicated that these differences show heterogeneity associated with the origin of the sample, indicating that the infection was caused by clones of different strains and that the polymorphism of DNA observed could make it difficult to choose one standard strain for an immune prophylactic treatment of glanders.

High frequency plantlet regeneration from rhizomatous buds in Mantisia spathulata Schult. and Mantisia wengeri Fischer and analysis of genetic uniformity using RAPD markers.

A protocol has been devised for enhanced in vitro regeneration of critically endangered Mantisia spathulata Schult. and Mantisia wengeri Fischer. Highest Bud Forming Capacity (BFC) of 6.10 +/- 0.55 with an average of 19.93 +/- 3.19 roots was obtained for M. spathulata within 5-6 weeks in Murashige and Skoogs (MS) medium supplemented with a combination of 10.0 microM of N6-benzyladenine (BA) and 2.5 microM of alpha-naphtalene acetic acid (NAA). For M. wengeri, BFC of 7.82 +/- 0.73 and 20.86 +/- 1.65 roots was achieved in MS media supplemented with a combination of 5.0 microM BA and 2.5 microM of NAA RAPD markers were used to evaluate the genetic stability of in vitro raised hardened plantlets. Similarity coefficient among the regenerated plants ranged between 0.85-0.98 for M. spathulata and 0.83-0.98 for M. wengeri. Maximum of 88 and 90% genetic similarity were obtained between in vitro raised hardened plantlets and mother stock of M. spathulata and M. wengeri, respectively through RAPD analysis. The hardened plantlets after RAPD analysis on being transferred to soil of experimental garden showed no marked phenotypic variations in vegetative or floral characteristics.

Molecular characterization of Malassezia sympodialis and Malassezia furfur from cattle with and without otitis / Caracterização molecular de isolados de Malassezia sympodialis e Malassezia furfur provenientes de bovinos com e sem otite externa

A molecular study of Malassezia strains isolated from cattle with or without otitis was carried out by random amplified polymorphic DNA analysis (RAPD). DNA was extracted and purified from nine strains of Malassezia sympodialis and fourteen of Malassezia furfur. These microorganisms were collected from eight different bovine herds in Minas Gerais state, Brazil. The RAPD analysis and phenograms did not show the formation of genetically distinct groups among the strain isolated from cattle with or without otitis raised in the same herds. Genetic heterogeneity was observed among Malassezia strains from different geographic origins. These data suggest that genetically similar M. sympodialis and M. furfur strains found as members of the normal ear microbiota could become opportunistically active in the inflammatory process in cattle.

A caracterização molecular de amostras de Malassezia spp., isoladas de bovinos com e sem otite, foi realizada por meio da técnica do DNA polimórfico amplificado ao acaso (RAPD). DNAs de nove amostras de Malassezia sympodialis e quatorze de M. furfur foram extraídos e purificados. Essas amostras foram provenientes de oito diferentes rebanhos bovinos no estado de Minas Gerais, Brasil. A análise de RAPD e os fenogramas não revelaram a formação de grupos geneticamente distintos entre amostras isoladas de bovinos, criados no mesmo rebanho, com ou sem otite. Heterogeneidade genética foi observada entre amostras de diferentes origens geográficas. Os dados sugerem que isolados geneticamente semelhantes e membros da microbiota normal do ouvido podem participar, como oportunistas, no processo inflamatório do conduto auditivo externo de bovinos.

Legionella in clinical specimens and hospital water supply facilities: molecular detection and genotyping of the isolates

To evaluate genus- and species-specific polymerase chain reactions [PCRs] for the detection of the genus Legionella and the species Legionella pneumophila in clinical specimens and hospital water supplies, and to establish a simple and reproducible random amplification of polymorphic DNA [RAPD]-PCR technique for genotyping of Legionella. A total of 70 respiratory tract specimens [bronchoalveolar lavage: n = 46; endotracheal secretions: n = 9; sputum: n = 15] from patients with atypical pneumonia, and 283 environmental samples [water: 20; swabs: 263] collected from water storage and supply facilities of the Mubarak Al-Kabeer Hospital, Kuwait, were tested by culture and genus-specific PCR for the detection of Legionella. The L pneumophila isolates were subsequently typed by serology and RAPD-PCR using serotype-specific sera and arbitrary primers, respectively. Of the 70 clinical samples, culture yielded 2 [2.9%] whereas genus-specific PCR detected Legionella in 20 [28.6%] samples. The 2 culture-positive specimens were also positive for L.-pneumophila-specific PCR. Testing of swab and water samples by culture and genus-specific PCR yielded 61 [21.6%] and 67 [23.7%] positive samples, respectively. All of the 61 culture-positive samples were also positive by genus-specific PCR and 45 of them were positive for L.-pneumophila-specific PCR. Serological typing of 43 L pneumophila isolates showed that 8 of these belonged to serotype 1 and 35 to serotype 3; however, RAPD-PCR analyses demonstrated polymorphisms among the isolates of both serotypes. A higher association between PCR and culture was observed for the environmental samples than for the clinical samples. The application of genus- and species-specific PCRs and RAPD is useful in the detection and typing of Legionella in clinical and environmental samples

Intra and inter populational genetic variability in Maytenus ilicifolia Mart. ex Reiss. 1861, through RAPD markers

Maytenus ilicifolia is a medicinal plant largely used in the South Brazilian folk medicine. The aim of this study was to quantify the intra and inter populational genetic variability in three populations of M. ilicifolia, focusing on the genetic conservation of this species, which has been threatened by anthropic action. RAPD (Random Amplified Polymorphic DNA) markers were used to analyze 30 plants of each of the three populations collected in the Alto Uruguai Gaúcho region. Fourteen selected primers generated a total of 158 bands, 71.5 percent of which were polymorphic. The comparison of Jaccard’s distances showed that the intra populational variation was higher than the inter populational variability, and cluster analysis allowed the separation of the three populations. Just 7.6 percent of the bands were specific of at least two populations. Data indicate that the analyzed M. ilicifolia populations represent a single genetic pool, and therefore any of the population thoroughly can represent the overall genetic variability of the species in the sampled region.

Maytenus ilicifolia é uma planta medicinal bastante utilizada na medicina popular da região sul do Brasil. O objetivo deste estudo foi quantificar a variabilidade genética intra e interpopulacional em três populações de M. ilicifolia visando a conservação genética desta espécie, que se encontra ameaçada pela ação antrópica. Marcadores RAPD (Random Amplified Polymorphic DNA) foram utilizados para analisar 30 plantas de cada uma das três populações coletadas na região do Alto Uruguai Gaúcho. Foram selecionados 14 primers, que geraram 158 bandas, das quais 71,5 por cento foram polimórficas. A comparação das distâncias de Jaccard mostraram que a variabilidade intra populacional foi maior que a interpopulacional, e a análise de agrupamentos permitiu a separação das três populações. Somente 7.6 por cento das bandas foram específicas de pelo menos duas populações. Os resultados indicam que as populações de M. ilicifolia analisadas representam um único conjunto gênico, de tal forma que qualquer uma das populações pode representar a variabilidade genética geral da espécie na região.

Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD) analysis / Caracterização genotípica de Haematobia irritans procedentes de diferentes regiões geográficas brasileiras baseada na análise do DNA polimórfico amplificado ao acaso (RAPD-PCR)

Blood-sucking diptera are important parasites in bovine production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.

Dípteras hematófagos são importantes parasitas dentro de sistemas de produção de bovinos, especialmente em confinamento. Haematobia irritans, a mosca-dos-chifres, é uma das espécies que maiores problemas causa em sistemas de produção de bovinos, dado ao intenso estresse que impõe aos animais. Um importante aspecto quando se estuda a variabilidade genética dentro das espécies é o estudo da estrutura geográfica destas populações. Buscando-se estimar a similaridade genotípica das diferentes populações brasileiras da mosca do chifre utilizou-se a técnica do DNA polimórfico amplificado ao acaso (RAPD-PCR), que mostrou-se eficiente para tal propósito. A utilização dos marcadores moleculares gerados através da técnica de RAPD-PCR tornou possível a identificação da origem geográfica das amostras das diferentes regiões geográficas brasileiras, assim como, estimar o fluxo genotípico entre as diferentes populações brasileiras da mosca-dos-chifres.