Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil / Determinação da heterogeneidade do genoma do vírus da leucose enzoótica bovina no estado de Santa Catarina, Brasil

Bovine leukemia virus (BLV) is a member of Retroviridae family, genus Deltaretrovirus, and the main viral agent responsible for economic loses in dairy herds. Some studies have been carried out about BLV genotypes, and at least seven genotypes were found out in samples of different regions of the world. The objective of this study was to identify BLV samples from seropositive dairy cattle in Santa Catarina state, Brazil, using molecular techniques. Blood samples were collected (454) from dairy cattle from 31 different farms, and serology using agar gel immunodiffusion test (AGID) was performed. After that, 191 seropositive samples were submitted to DNA extraction, and in 77 samples the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene was performed. Nineteen DNA samples were subjected to restriction fragment length polymorphism (RFLP) analysis by digestion of the PCR fragment by five restriction endonucleases - BamHI, HaeIII, Tru9I, TaqI, and MwoI. It was found 42% seropositive animals (191/454) and 68% positives of the farms (21/31). The PCR showed 80.5% (62/77) of animals positive. The RFLP analysis identified five different genotypes dispersed by Santa Catarina state, with the highest prevalence for genotype X (47.4%). Overall, our results identified the viral genotypes present in dairy cattle and the prevalence of new variants in representative farms from Santa Catarina state.(AU)

O bovine leukemia virus (BLV) é um membro da família Retroviridae, gênero Deltaretrovirus, e o principal agente viral causador de perdas econômicas em rebanhos leiteiros. Diversos estudos têm sido feitos sobre os genótipos de BLV, e foram encontrados pelo menos sete em amostras de diferentes partes do mundo. O objetivo deste estudo foi realizar a caracterização molecular de amostras de BLV de bovinos leiteiros soropositivos no estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos de 31 propriedades, e fez-se inicialmente a sorologia por meio do teste de imunodifusão em gel de ágar. Após a sorologia, 191 amostras soropositivas foram então submetidas à extração de DNA, e em 77 amostras se realizou a reação da polimerase em cadeia (PCR), para a amplificação de um fragmento de 440 pb do gene env. Dezenove amostras foram submetidas à análise do polimorfismo dos fragmentos de restrição por digestão do fragmento da PCR por cinco enzimas de restrição: BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia apontaram 42% de animais soropositivos (191/454) e 68% de propriedades positivas (21/31). Na PCR, 80,52% (62/77) dos animais apresentaram-se positivos. A análise do polimorfismo dos fragmentos de restrição identificou cinco genótipos circulantes no estado, e a maior prevalência foi observada no genótipo X (47,4%). Este estudo permite-nos conhecer alguns dos genótipos virais presentes em bovinos leiteiros do estado de Santa Catarina, bem como identificar a existência de novas variantes e sua prevalência atual, e os resultados são úteis para futuros estudos epidemiológicos.(AU)

Heterogeneity determination of bovine leukemia virus genome in Santa Catarina state, Brazil / Determinação da heterogeneidade do genoma do vírus da leucose enzoótica bovina no estado de Santa Catarina, Brasil

Bovine leukemia virus (BLV) is a member of Retroviridae family, genus Deltaretrovirus, and the main viral agent responsible for economic loses in dairy herds. Some studies have been carried out about BLV genotypes, and at least seven genotypes were found out in samples of different regions of the world. The objective of this study was to identify BLV samples from seropositive dairy cattle in Santa Catarina state, Brazil, using molecular techniques. Blood samples were collected (454) from dairy cattle from 31 different farms, and serology using agar gel immunodiffusion test (AGID) was performed. After that, 191 seropositive samples were submitted to DNA extraction, and in 77 samples the polymerase chain reaction (PCR) for amplification of a 440 bp fragment of the env gene was performed. Nineteen DNA samples were subjected to restriction fragment length polymorphism (RFLP) analysis by digestion of the PCR fragment by five restriction endonucleases - BamHI, HaeIII, Tru9I, TaqI, and MwoI. It was found 42% seropositive animals (191/454) and 68% positives of the farms (21/31). The PCR showed 80.5% (62/77) of animals positive. The RFLP analysis identified five different genotypes dispersed by Santa Catarina state, with the highest prevalence for genotype X (47.4%). Overall, our results identified the viral genotypes present in dairy cattle and the prevalence of new variants in representative farms from Santa Catarina state.(AU)

O bovine leukemia virus (BLV) é um membro da família Retroviridae, gênero Deltaretrovirus, e o principal agente viral causador de perdas econômicas em rebanhos leiteiros. Diversos estudos têm sido feitos sobre os genótipos de BLV, e foram encontrados pelo menos sete em amostras de diferentes partes do mundo. O objetivo deste estudo foi realizar a caracterização molecular de amostras de BLV de bovinos leiteiros soropositivos no estado de Santa Catarina. Foram coletadas 454 amostras de sangue de bovinos de 31 propriedades, e fez-se inicialmente a sorologia por meio do teste de imunodifusão em gel de ágar. Após a sorologia, 191 amostras soropositivas foram então submetidas à extração de DNA, e em 77 amostras se realizou a reação da polimerase em cadeia (PCR), para a amplificação de um fragmento de 440 pb do gene env. Dezenove amostras foram submetidas à análise do polimorfismo dos fragmentos de restrição por digestão do fragmento da PCR por cinco enzimas de restrição: BamHI, HaeIII, Tru9I, TaqI e MwoI. Os resultados obtidos na sorologia apontaram 42% de animais soropositivos (191/454) e 68% de propriedades positivas (21/31). Na PCR, 80,52% (62/77) dos animais apresentaram-se positivos. A análise do polimorfismo dos fragmentos de restrição identificou cinco genótipos circulantes no estado, e a maior prevalência foi observada no genótipo X (47,4%). Este estudo permite-nos conhecer alguns dos genótipos virais presentes em bovinos leiteiros do estado de Santa Catarina, bem como identificar a existência de novas variantes e sua prevalência atual, e os resultados são úteis para futuros estudos epidemiológicos.(AU)

Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high purity DNA from soil microbiota, five prewashing agents were compared in terms of their efficiency and effectiveness in removing soil contaminants. Residual contaminants were precipitated by adding 0.6 mL of 0.5 M CaCl2. Four cell lysis methods were applied to test their compatibility with the pretreatment (prewashing + Ca2+ flocculation) and to ultimately identify the optimal cell lysis method for analyzing fungal communities in forest soils. The results showed that pretreatment with TNP + Triton X-100 + skim milk (100 mM Tris, 100 mM Na4P2O7, 1% polyvinylpyrrolidone, 100 mM NaCl, 0.05% Triton X-100, 4% skim milk, pH 10.0) removed most soil humic contaminants. When the pretreatment was combined with Ca2+ flocculation, the purity of all soil DNA samples was further improved. DNA samples obtained by the fast glass bead-beating method (MethodFGB) had the highest purity. The resulting DNA was successfully used, without further purification steps, as a template for polymerase chain reaction targeting fungal internal transcribed spacer regions. The results obtained by terminal restriction fragment length polymorphism analysis indicated that the MethodFGB revealed greater fungal diversity and more distinctive community structure compared with the other methods tested. Our study provides a protocol for fungal cell lysis in soil, which is fast, convenient, and effective for analyzing fungal communities in forest soils.(AU)

A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida dubliniensis

Candida dubliniensis is an emerging pathogen capable of causing superficial as well as systemic infections. Due to its close similarity to C. albcians, conventional methods based on phenotypic traits are not always reliable in identification of C. dubliniensis. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay to identify and discriminate between the two closely related species. The D1/D2 region of 28S rDNA was amplified by PCR and enzymatically digested by ApaI and BsiEI respectively. PCR products of both species were digested into two fragments by ApaI, but those of other yeast species were undigested. BsiEI cut the PCR products of C. albicans into two fragments but not those of C. dubliniensis. Thus two species were differentiated. We evaluated 10 reference strains representing 10 yeast species, among which C. albicans and C. dubliniensis were successfully identified. A total of 56 phenotypically characterized clinical isolates (42 C. albicans isolates and 14 C. dubliniensis isolates) were also investigated for intra-species variability. All tested isolates produced identical RFLP patterns to their respective reference strains except one initially misidentified isolate. Our method offers a simple, rapid and reliable molecular method for the identification of C. albicans and C. dubliniensis.

Quantitative analysis of Terminal Restriction Fragment Length Polymorphism (T-RFLP) microbial community profiles: peak height data showed to be more reproducible than peak area

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a culture-independent fingerprinting method for microbial community analysis. Profiles generated by an automated electrophoresis system can be analysed quantitatively using either peak height or peak area data. Statistical testing demontrated that peak height data showed to be more reproducible than peak area data.

Terminal Restriction Fragment Length Polymorphism (T-RFLP) é um método molecular, independente de cultivo, para análise de comunidades microbianas. Perfis gerados por um sistema automatizado de eletroforese podem ser analisados quantitativamente usando dados de altura ou área dos picos. Os dados de altura mostraram-se mais reprodutíveis do que os de área.