Marcadores moleculares para la taxonomía e identificación del género Brucella (Alphaproteobacteria) / Molecular markers for the taxonomy and identification of the genus Brucella (Alphaproteobacteria)

Introducción: El género Brucella está incluido en la familia Brucellaceae que pertenece al orden Rhizobiales y es reconocido por su alto grado de patogenicidad. Las bacterias de este género son responsables de la brucelosis, enfermedad que ha sido reportada como una de las zoonosis más importantes a nivel mundial por su incidencia en el ganado y el hombre. Los estudios previos para la clasificación taxonómica del género, se han basado fundamentalmente en el análisis del gen 16S ARNr. Sin embargo, pocas investigaciones se han dirigido a la identificación de marcadores moleculares que distingan a sus miembros de otros grupos de bacterias. Objetivo: Identificar inserciones en secuencias de proteínas conservadas, que pudieran ser utilizados como marcadores moleculares para la taxonomía y diagnóstico de especies del género Brucella. Métodos: Las secuencias homólogas de las proteínas analizadas fueron obtenidas de bases de datos internacionales y, posteriormente, alineadas con el programa ClustalX2, para ello fueron considerados los parámetros sugeridos en la literatura. Resultados: Se identificaron inserciones en las proteínas oxoglutarato deshidrogenasa (componente E1) y ADN ligasa A específicas del género Brucella. Conclusiones: Las inserciones halladas pueden ser empleadas como complemento a los métodos tradicionales de clasificación taxonómica y para el diagnóstico molecular de bacterias incluidas en el género Brucella(AU)

Introduction: Brucella is a genus from the Brucellaceae family, Rhizobiales order. This genus is recognized for its high pathogenicity. Brucella bacteria cause brucellosis, a disease reported as one of the most important zoonoses worldwide due to its incidence in cattle and people. Previous studies on taxonomic classification of the genus have been mainly based on the analysis of gene 16S rDNA. However, few studies have been aimed at identification of molecular markers distinguishing its members from other groups of bacteria. Objective: Identify insertions in preserved protein sequences which could be used as molecular markers for the taxonomy and diagnosis of species from the Brucella genus. Methods: The homologous sequences for the proteins analyzed were obtained from international databases and aligned with the software ClustalX2, considering the parameters suggested in the literature. Results: Insertions were identified in the proteins oxoglutarate dehydrogenase (component E1) and DNA ligase A, specific of the genus Brucella. Conclusions: The insertions found may be used as complements to the traditional methods for taxonomic classification and for the molecular diagnosis of bacteria from the genus Brucella(AU)

Two Compound Heterozygous Were Identified in SLC26A4 Gene in Two Chinese Families With Enlarged Vestibular Aqueduct

OBJECTIVES: To investigate the genetic causes of hearing loss with enlarged vestibular aqueduct (EVA) in two children from unrelated two Chinese families. METHODS: Sanger sequencing of all coding exons in SLC26A4 (encoding Pendrin protein) was performed on the two patients, their sibling and parents respectively. To predict and visualize the potential functional outcome of the novel variant, model building, structure analysis, and in silico analysis were further conducted. RESULTS: The results showed that the proband from family I harbored a compound heterozygote of SLC26A4 c.1174A>T (p.N392Y) mutation and c.1181delTCT (p.F394del) variant in exon 10, potentially altering Pendrin protein structure. In family II, the proband was identified in compound heterozygosity with a known mutation of c.919-2A>G in the splice site of intron 7 and a novel mutation of c.1023insC in exon 9, which results in a frameshift and translational termination, consequently leading to truncated Pendrin protein. Sequence homology analysis indicated that all the mutations localized at high conservation sites, which emphasized the significance of these mutations on Pendrin spatial organization and function. CONCLUSION: In summary, this study revealed two compound heterozygous mutations (c.1174A>T/c.1181delTCT; c.919- 2A>G/c.1023insC) in Pendrin protein, which might account for the deafness of the two probands clinically diagnosed with EVA. Thus this study contributes to improve understanding of the causes of hearing loss associated with EVA and develop a more scientific screening strategy for deafness.

Blood transcriptome resources of chinstrap (Pygoscelis antarcticus) and gentoo (Pygoscelis papua) penguins from the South Shetland Islands, Antarctica

The chinstrap (Pygoscelis antarcticus) and gentoo (P. papua) penguins are distributed throughout Antarctica and the sub-Antarctic islands. In this study, high-quality de novo assemblies of blood transcriptomes from these penguins were generated using the Illumina MiSeq platform. A total of 22.2 and 21.8 raw reads were obtained from chinstrap and gentoo penguins, respectively. These reads were assembled using the Oases assembly platform and resulted in 26,036 and 21,854 contigs with N50 values of 929 and 933 base pairs, respectively. Functional gene annotations through pathway analyses of the Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed for each blood transcriptome, resulting in a similar compositional order between the two transcriptomes. Ortholog comparisons with previously published transcriptomes from the Adélie (P. adeliae) and emperor (Aptenodytes forsteri) penguins revealed that a high proportion of the four penguins’ transcriptomes had significant sequence homology. Because blood and tissues of penguins have been used to monitor pollution in Antarctica, immune parameters in blood could be important indicators for understanding the health status of penguins and other Antarctic animals. In the blood transcriptomes, KEGG analyses detected many essential genes involved in the major innate immunity pathways, which are key metabolic pathways for maintaining homeostasis against exogenous infections or toxins. Blood transcriptome studies such as this may be useful for checking the immune and health status of penguins without sacrifice.

Inducible cellulase production from an organic solvent tolerant Bacillus sp. SV1 and evolutionary divergence of endoglucanase in different species of the genus Bacillus

Abstract Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and β-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times β-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII. 4 2012" variants in UK coastal waters during 2013

Abstract This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks.

High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates

Abstract Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70 nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113 nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

Malignant Catarrhal Fever in Brazilian cattle presenting with neurological syndrome

Abstract Malignant Catarrhal Fever (MCF) was investigated in the central nervous system of cattle with neurological syndrome. Two-hundred-ninety samples were analyzed by histology, and molecular methods to detect ovine herpesvirus type 2 (OvHV-2) were optimized and validated. The qualitative polymerase chain reaction (qualitative PCR) analytical sensitivity was 101 DNA copies/µL and found 4.8% (14/290) positive for OvHV-2. The quantitative polymerase chain reaction (qPCR) analytical sensitivity was 100 DNA copy/µL and 5.9% (17/290) positivity, with 47.1% (8/17) of the positive samples presenting histological evidence of non-purulent meningo-encephalitis. The qualitative PCR products (422 bp of the ORF75 region) were sequenced and submitted to phylogenetic analysis. Identity matrices showed 100% similarity in OvHV-2 samples obtained in this study and those recovered from GenBank, corroborating other studies.

Evaluation of prevalence, homology and immunogenicity of dispersin among enteroaggregative Escherichia coli isolates from Iran

Background: Diarrhea, caused by enteroaggregative Escherichia coli [EAEC], is an important infection leading toillness and death. Numerous virulent factors have been described in EAEC. However, their prevalence was highly variable among EAECs of distinct geographic locations. Studies have shown that dispersin [antiaggregation protein, aap] is one of the important and abundant virulent factors in EAEC. In this study, we aimed to determine the presence, conservation, and immunogenicity of aap gene in EAEC isolated from Iranian patients

Methods: PCR amplification of aap gene in the EAEC isolates was performed, and the aap gene was cloned in pBAD-gIIIA vector. The sequence of aap gene was analyzed using the ExPASy and BLAST tools. The expression of aap gene was performed in E. coli Top10, and expression confirmation was carried out by SDS-PAGE and Western-blot techniques. Rabbits were immunized with purified dispersin protein emulsified with Freund's adjuvant. Sera were collected and examined for antibody response. Finally, in vitro efficacy of dispersin and anti-dispersin was evaluated

Results: The results of PCR showed the presence of aap gene in all of the EAEC isolates with significant homology. Finally, the significant difference between the levels of IgG response in dispersin-injected rabbits and control group was observed

Conclusion: Our results were in accordance with other studies that reported the presence of dispersin in the EAEC isolates with high conservation and immunogenicity. Hence, dispersin could be a promising candidate for any probable prevention against EAEC infections

Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India

Abstract Mercury, which is ubiquitous and recalcitrant to biodegradation processes, threatens human health by escaping to the environment via various natural and anthropogenic activities. Non-biodegradability of mercury pollutants has necessitated the development and implementation of economic alternatives with promising potential to remove metals from the environment. Enhancement of microbial based remediation strategies through genetic engineering approaches provides one such alternative with a promising future. In this study, bacterial isolates inhabiting polluted sites were screened for tolerance to varying concentrations of mercuric chloride. Following identification, several Pseudomonas and Klebsiella species were found to exhibit the highest tolerance to both organic and inorganic mercury. Screened bacterial isolates were examined for their genetic make-up in terms of the presence of genes (merP and merT) involved in the transport of mercury across the membrane either alone or in combination to deal with the toxic mercury. Gene sequence analysis revealed that the merP gene showed 86–99% homology, while the merT gene showed >98% homology with previously reported sequences. By exploring the genes involved in imparting metal resistance to bacteria, this study will serve to highlight the credentials that are particularly advantageous for their practical application to remediation of mercury from the environment.

Shellfish and House Dust Mite Allergies: Is the Link Tropomyosin?

Crustacean shellfish allergy is an important cause of food allergy and anaphylaxis in Asia. The major allergen in shellfish allergy is tropomyosin, a pan-allergen that is also found in house dust mites and cockroaches. Tropomyosins from house dust mites (HDMs) have a high sequence homology to shellfish tropomyosins, and cross-reactivity between HDM and shrimp tropomyosins has been demonstrated. Exposure to inhaled tropomyosins from house dust mites has been postulated to be the primary sensitizer for shellfish allergy, in a reaction analogous to the oral allergy (inhalant-food) syndrome. This notion is supported by indirect data from the effects of HDM immunotherapy on shellfish allergy, and strong correlations of shellfish and HDM sensitization. HDM immunotherapy has been reported to induce both shrimp allergy in non-allergic patients and shrimp tolerance in shrimp-allergic patients. Epidemiological surveys have also demonstrated a strong correlation between shellfish and HDM sensitization in both hospital-based and community-based studies. Unexposed populations have also been shown to develop sensitization-shellfish sensitization in orthodox Jews with no history of shellfish consumption was associated with HDM sensitization. Reciprocally, HDM sensitization in an Icelandic population living in a HDM-free environment was associated with shrimp sensitization. In vitro IgE inhibition studies on sera in shrimp-allergic Spanish patients indicate that mites are the primary sensitizer in shrimp-allergic patients living in humid and warm climates. Current data supports the hypothesis that tropomyosin is the link between HDM and shellfish allergies. The role of tropomyosin in HDM and shellfish allergies is a fertile field for investigation as it may provide novel immunotherapeutic strategies for shellfish allergy.

Seroepidemiological Survey of Zoonotic Diseases in Small Mammals with PCR Detection of Orientia tsutsugamushi in Chiggers, Gwangju, Korea

Serosurveillance for zoonotic diseases in small mammals and detection of chiggers, the vector of Orientia tsutsugamushi, were conducted from September 2014 to August 2015 in Gwangju Metropolitan Area. Apodemus agrarius was the most commonly collected small mammals (158; 91.8%), followed by Myodes regulus (8; 4.6%), and Crocidura lasiura (6; 3.5%). The highest seroprevalence of small mammals for O. tsutsugamushi (41; 26.3%) was followed by hantaviruses (24; 15.4%), Rickettsia spp. (22; 14.1%), and Leptospira (2; 1.3%). A total of 3,194 chiggers were collected from small mammals, and 1,236 of 3,194 chiggers were identified with 7 species of 3 genera: Leptotrombidium scutellare was the most commonly collected species (585; 47.3%), followed by L. orientale (422; 34.1%), Euchoengastia koreaensis (99; 8.0%), L. palpale (58; 4.7%), L. pallidum (36; 2.9%), Neotrombicula gardellai (28; 2.3%), and L. zetum (8; 0.6%). L. scutellare was the predominant species. Three of 1,236 chigger mites were positive for O. tsutsugamushi by PCR. As a result of phylogenetic analysis, the O. tsutsugamushi strain of chigger mites had sequence homology of 90.1-98.2% with Boryong. This study provides baseline data on the distribution of zoonotic diseases and potential vectors for the development of prevention strategies of vector borne diseases in Gwangju metropolitan area.

Early surgical complications after gastric by-pass: a literature review / Complicações cirúrgicas precoces após bypass gástrico: revisão da literatura

INTRODUCTION: Gastric bypass is today the most frequently performed bariatric procedure,but, despite of it, several complications can occur with varied morbimortality. Probably all bariatric surgeons know these complications, but, as bariatric surgery continues to spread, general surgeon must be familiarized to it and its management. Gastric bypass complications can be divided into two groups: early and late complications, taking into account the two weeks period after the surgery. This paper will focus the early ones. METHOD: Literature review was carried out using Medline/PubMed, Cochrane Library, SciELO, and additional information on institutional sites of interest crossing the headings: gastric bypass AND complications; follow-up studies AND complications; postoperative complications AND anastomosis, Roux-en-Y; obesity AND postoperative complications. Search language was English. RESULTS: There were selected 26 studies that matched the headings. Early complications included: anastomotic or staple line leaks, gastrointestinal bleeding, intestinal obstruction and incorrect Roux limb reconstruction. CONCLUSION: Knowledge on strategies on how to reduce the risk and incidence of complications must be acquired, and every surgeon must be familiar with these complications in order to achieve an earlier recognition and perform the best intervention. .

INTRODUÇÃO: O bypass gástrico é hoje o procedimento bariátrico mais realizado, mas, apesar disso, várias complicações podem ocorrer com variada morbimortalidade. Provavelmente todos os cirurgiões bariátricos conhecem essas complicações, mas como a cirurgia bariátrica continua a se espalhar, o cirurgião geral deve estar familiarizado com essas complicações e seu manuseio. As complicações do bypass gástrico podem ser divididas em dois grupos: as precoces e tardias, tendo em conta o período de duas semanas após a operação. Este artigo irá focar as precoces. MÉTODO: Foi realizada revisão da literatura utilizando as bases Medline/PubMed, Cochrane Library, SciELO, e informações adicionais sobre sites institucionais de interesse cruzando os descritores: bypass gástrico AND complicações; seguimento AND complicações; complicações pós-operatórias AND anastomose, Roux-en-Y; obesidade AND complicações pós-operatórias. A língua usada para a busca foi o inglês. RESULTADOS: Foram selecionados 26 artigos que combinavam com os descritores. As complicações imediatas foram: fístula na linha de grampeamento, sangramento gastrointestinal, obstrução intestinal e reconstrução incorreta da alça em Roux. CONCLUSÃO: O conhecimento sobre as estratégias de como reduzir o risco e incidência das complicações deve ser adquirido ao longo do tempo, e cada cirurgião deve estar familiarizado com essas complicações, a fim de reconhecê-las precocemente e realizar a melhor intervenção. .

Four Additional Cases of Diphyllobothrium nihonkaiense Infection Confirmed by Analysis of COX1 Gene in Korea

Most of the diphyllobothriid tapeworms isolated from human samples in the Republic of Korea (= Korea) have been identified as Diphyllobothrium nihonkaiense by genetic analysis. This paper reports confirmation of D. nihonkaiense infections in 4 additional human samples obtained between 1995 and 2014, which were analyzed at the Department of Parasitology, Hallym University College of Medicine, Korea. Analysis of the mitochondrial cytochrome c oxidase 1 (cox1) gene revealed a 98.5-99.5% similarity with a reference D. nihonkaiense sequence in GenBank. The present report adds 4 cases of D. nihonkaiense infections to the literature, indicating that the dominant diphyllobothriid tapeworm species in Korea is D. nihonkaiense but not D. latum.

Four Additional Cases of Diphyllobothrium nihonkaiense Infection Confirmed by Analysis of COX1 Gene in Korea

Most of the diphyllobothriid tapeworms isolated from human samples in the Republic of Korea (= Korea) have been identified as Diphyllobothrium nihonkaiense by genetic analysis. This paper reports confirmation of D. nihonkaiense infections in 4 additional human samples obtained between 1995 and 2014, which were analyzed at the Department of Parasitology, Hallym University College of Medicine, Korea. Analysis of the mitochondrial cytochrome c oxidase 1 (cox1) gene revealed a 98.5-99.5% similarity with a reference D. nihonkaiense sequence in GenBank. The present report adds 4 cases of D. nihonkaiense infections to the literature, indicating that the dominant diphyllobothriid tapeworm species in Korea is D. nihonkaiense but not D. latum.

Allergenic Characterization of a Novel Allergen, Homologous to Chymotrypsin, from German Cockroach

PURPOSE: Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease. METHODS: A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA. RESULTS: The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract. CONCLUSIONS: A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.

Isolation and identification of the Akabane virus from mosquitoes in Yunnan Province, China / 病毒学报

To evaluate the prevalence of mosquito-borne viruses in Manshi and Ruili (Yunnan Province, China), we collected 2 149 mosquitoes (17 species) in August 2010. Virus isolation was undertaken by the cul- ture of baby hamster kidney cells (BHK-21 cells). Two virus-like isolates were obtained: DHL10M117 was isolated from collected in Mangshi; DHL10M110 was obtained from Anopheles vagus collected in Rui- li. Both isolates caused cytopathic effects,illness and death in suckling mice inoculated with these isolates via the intracerebral route. Two positive amplicons, 702-bp from the S segment and 456-bp from the M segment,were obtained using reverse transcription-polymerase chain reaction using primers specific for the Akabane virus (AKV). Phylogenetic analysis suggested that these two virus stains had a distant relation- ship with AKVs from Kenya and Australia,but were genetically close to those from Japan,South Korea, and Taiwan. However,they were separate from other Asian strains and grouped into a small branch. The highest nucleotide and amino-acid sequence identity of the S segment was found with the CY-77 strain from Taiwan (96.6% and 99.6% for DHL10M117 and 96.7% and 100% for DHL10M110,respectively). Com- parison of the M segment showed they shared the highest amino acid identity with CY-77 (99.6% and 100%, respectively), whereas the highest nucleotide identity was found with the Iriki strain from Japan (99.6% and 100%, respectively). Compared with the MP496 strain from Kenya,they displayed lower lev- els of sequence homology, at 69.7% and 70.0% for nucleotide sequences of the two loci,and 91. 0% for a- mino acids. Our results identified that DHL10M117 and DHL10M110 were strains of AKV,and provided molecular biological evidence for the existence of AKV in Yunnan Province. These AKV strains that are circulating in Yunnan Province share a close genetic relationship with strains from the rest of Asia. Culex tritaeniorhynchus and Anopheles vagus may serve as transmission vectors.

Genetic Characteristics of Echovirus Type 6 Isolated from Hunan Province, China, 2009-2014 / 病毒学报

We wished to understand the genetic characteristics of enteric cytopathic human orphan (ECHO) virus type 6 (ECHO6) circulating in China. First, the partial VP1 coding region of six strains of the ECH-O6 virus isolated from cases of hand, foot and mouth diseases during routine surveillance in Hunan Province (China) from 2009 to 2014 were sequenced. Those sequences were analyzed along with 138 sequences of ECHO viruses covering five provinces of China and countries outside China retrieved from the GenBank database. A phylogenetic tree based on partial VPI was constructed, and it indicated that Chinese strains of the ECHO virus could form two distinct evolutionary branches: branch 1 and branch 2. All isolates of the ECHO virus from Hunan Province belonged to the 2c subranch, which revealed that they may share a common evolutionary origin. ECHO strains in branch 2 may be the predominant strains in China due to their wide geographic distribution and long period of circulation. We used nucleotide differences of >30%o as the basis of cluster division. ECHO, viruses could be divided into four clusters (A-D). Cluster D could be divided further into ten subclusters on the basis of nucleotide differences of 15%-30%. All ECHO6 isolates from Hunan Province belonged to the D7 subcluster. These data showed that the ECHO6 strains that circulated in Hunan Province in 2009-2014 were closely related to each other, and probably shared a common evolutionary origin. In addition, at least four distinct lineages of ECHO viruses have circulated in China.

Rotavirus genotypes and the indigenous children of Brazilian midwest in the vaccine era, 2008-2012: footprints of animal genome

World group A rotavirus (RVA) surveillance data provides useful estimates of the disease burden, however, indigenous population might require special consideration. The aim of this study was to describe the results of G­ and P­types from Brazilian native children ≤3 years. Furthermore, selected strains have been analyzed for the VP7, VP6, VP4, and NSP4 encoding genes in order to gain insight into genetic variability of Brazilian strains. A total of 149 samples, collected during 2008­2012, were tested for RVA using ELISA and PAGE, following by RT­PCR and sequencing. RVA infection was detected in 8.7% of samples (13/149). Genotype G2P[4] was detected in 2008 and 2010, G8P[6] in 2009, and G3P[8] in 2011. The phylogenetic analysis of the VP7 and VP4 genes grouped the Brazilian G2P[4] and G3P[8] strains within the lineages currently circulating in humans worldwide. However, the phylogenetic analysis of the VP6 and NSP4 from the Brazilian G2P[4] strains, and the VP7 and NSP4 from the Brazilian G3P[8] strains suggest a distant common ancestor with different animal strains (bovine, caprine, and porcine). The epidemiological and genetic information obtained in the present study is expected to provide an updated understanding of RVA genotypes circulating in the native infant population, and to formulate policies for the use of RVA vaccines in indigenous Brazilian people. Moreover, these results highlight the great diversity of human RVA strains circulating in Brazil, and an in­depth surveillance of human and animal RVA will lead to a better understanding of the complex dynamics of RVA evolution

Molecular characterization of the first leptospires isolated from goats in Brazil

Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Identificación y expresión diferencial del factor de transcripción asociado a microftalmia en corazón y cardiomiocitos aislados de cobayo: su posible papel en la hipertrofia y la viabilidad / Identification and differential expression of the microphthalmia-associated transcription factor in heart and isolated cardiomyocytes from Guinea pigs: Possible role in hypertrophy and viability

Introducción. El factor de transcripción asociado a la microftalmia ( Microphtalmia-Associated Transcription Factor , MITF) regula la expresión de genes específicos, pero no se conoce su expresión y su función a nivel cardiaco. Objetivos. Identificar la expresión del MITF en corazón y en cardiomiocitos aislados de cobayo, describir los cambios morfológicos asociados con su disminución y evaluar los niveles relativos de su expresión en cardiomiocitos aislados en condiciones de preacondicionamiento isquémico. Materiales y métodos. El análisis de la expresión relativa de la isoforma específica de tejido cardiaco ( heart-type MITF, MITF-H), se determinó mediante reacción en cadena de la polimerasa (PCR) en tiempo real semicuantitativa, secuenciación y Western blot . La disminución del ARNm del MITF se indujo con un ARN pequeño de interferencia ( short hairpin RNA interference , shRNAi) específico. El tamaño, el diámetro y el número de fibras musculares se evaluaron por observación directa con microscopía de luz. Resultados. Se amplificó un fragmento de 281 pb de ADNc; el análisis de la secuencia confirmó la identidad del exón 1 y la isoforma H del MITF. La interferencia del ARNm del MITF se asoció con un mayor índice cardiaco (peso corazón/peso corporal: 5,46 x 10 -3 Vs. 4,6 x 10 -3 ) y un incremento del diámetro de las fibras cardiacas (50,2±16 µm Vs. 38,7±14,7 µm; p<0,05, n=150). En los cardiomiocitos aislados en condiciones de preacondicionamiento isquémico, se observó una expresión relativa del MITF-H mayor que en los miocitos en normoxia y expuestos a lesión por isquemia simulada (80 y 100 veces más, n=5, p<0,05, n=3). Conclusión. Los resultados sugieren que el MITF-H podría estar involucrado en la hipertrofia, la respuesta al estrés por isquemia y la supervivencia de cardiomiocitos de cobayo.

Introduction: The microphthalmia -associated transcription factor ( MITF ) regulates the expression of specific genes and its cardiac expression and function is not known. Objectives: To identify the expression of MITF in hearts and isolated cardiomyocytes from Guinea pigs, to describe morphological changes associated with mRNA interference of MITF and to evaluate their relative changes in expression in isolated cardiomyocytes under ischemic preconditioning. Materials and methods: The cardiac specific isoform, MITF-H, and relative expression level analysis, was determined by semi-quantitative real time PCR, sequencing and Western blotting. Reduction of mRNA-MITF-H was induced by transduction of specific-MITF-shRNAi interference. The cardiac morphological changes, diameter and number of cardiac fibers were evaluated by direct observation and light microscopy. Results: A cDNA fragment of 281 bp was amplified from heart and isolated ventricular cardiac myocytes. Sequence analysis confirmed the identity of the isoform MITF-H, exon 1. The MITF silencing was associated with an increase in cardiac index (heart weight/body weight vs . 5.46 x 10 -3 vs 4.6 x 10 -3 ) and higher diameter of cardiac fibers (50.2±16 µ m vs 38,7±14,7 µ m p<0.05, n=150). In isolated cardiac myocytes under ischemic preconditioning we observed a higher relative expression compared with that measured in myocytes exposed to normoxia and simulated ischemia (eighty and one hundred times, p <0.05, n = 5). Conclusion. The results suggest that MITF-H isoform may be involved in Guinea pig cardiac hypertrophy, response to stress by ischemia and cardiomyocytes survival.