Cytochrome-b variation in Apis mellifera samples and its association with COI-COII patterns

Five Mbo I (Mbo-A, Mbo-M, Mbo-C(1), Mbo-C(2) and Mbo-C(3)) and Hinf I (Hinf-1 to Hinf-5) patterns were observed in Apis mellifera samples after restriction of a 485 bp fragment of the mitochondrial cytochrome-b (cyt-b) gene. Associating the cyt-b Restriction fragment length polymorphism (RFLP) pattern of each sample to its respective previously established COI-COII (Dra I sites) pattern, five restriction patterns (Mbo-C(1), Mbo-C(2), Mbo-C(3), Hinf-1 and Hinf-4) were observed in samples of maternal origin associated to the evolutionary branch C. No deletions or insertions were observed and the nucleotide substitution rate was estimated at 5.4%. Higher nucleotide diversity was observed among the branch C-haplotypes when compared with A and M lineages. Further studies are needed to confirm if the cyt-b + COI-COII haplotypes help to assign certain phylogeographic patterns to the branch C and to clarify phylogenetic relationships among A. mellifera subspecies.

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the eft operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the eft operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in Sao Paulo, Brazil

Aims: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. Methods and Results: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. Conclusions: This study demonstrated that healthy sheep in Sao Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. Significance and Impact of the Study: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Epidemiologia molecular da tuberculose resistente a múltiplos fármacos no Rio Grande do Sul

A tuberculose resistente a múltiplos fármacos (TB MDR) é definida como uma forma de tuberculose (TB) causada por Mycobacterium tuberculosis resistente a pelo menos isoniazida e rifampicina. A TB MDR é um problema mundial crescente resultante da não adesão dos pacientes ao tratamento e pelo gerenciamento ineficaz da doença pelos sistemas de saúde. Este estudo foi realizado com o objetivo de identificar os fatores de risco e os padrões de transmissão da TB MDR no Estado do Rio Grande do Sul, comparando os resultados obtidos com aqueles casos de TB suscetíveis aos fármacos. Durante os anos de 1999 e 2000 foram identificados 60 isolados MDR no Laboratório Central do RS (LACEN) e 202 isolados suscetíveis aos fármacos anti-TB. Estes isolados foram analisados utilizando a técnica de Polimorfismo do Tamanho dos Fragmentos de Restrição (RFLP) baseado no IS6110. Os dados clínicos e demográficos dos pacientes portadores destas linhagens também foram analisados. Nos isolados que apresentaram seis ou menos cópias de IS6110 foi realizada uma segunda técnica de genotipagem, o Spoligotyping. Os pacientes portadores de linhagens de M. tuberculosis com padrões idênticos foram considerados clusters. Foi observado que entre os 262 isolados, 94 (36%) pertenciam a 20 distintos clusters, e após a análise por Spoligotyping, 89 destes isolados (34%) permaneceram em cluster. Os isolados MDR não diferiram estatisticamente dos isolados suscetíveis na proporção de formação de cluster. Foi observada associação significante entre a ocorrência de TB MDR e tratamento prévio (p < 0,001) e falência no tratamento (p < 0,001). No entanto, os pacientes HIV positivos foram associados com TB suscetível (p = 0,024). Também foi identificado que pacientes não casados desenvolveram mais TB devida à transmissão recente (p < 0,005). A introdução da terapia supervisionada de curta duração (DOTS) no RS será importante, pois auxiliará na diminuição das taxas de falência e abandono de tratamento, evitando o desenvolvimento de novas linhagens MDR.

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterações nos genes da E-caderina e β-catenina em adenoma pleomórfico e carcinoma adenóide cístico: estudo molecular e imuno-histoquímico

Pleomorphic adenoma and adenoid cystic carcinoma represent a benign and malignant salivary gland neoplasm, respectively, that shares the same histological origin, however with distinct biological behavior. The aim of the present study was identify the -160 C/A polymorphism in the gene CDH1, mutational analysis of CTNNB1 gene and evaluation the expression of the E-cadherin and β-catenin in pleomorphic adenomas and adenoid cystic carcinomas. Furthermore, it was proposed correlate the immunochemistry staining patterns with the polymorphism and mutations. Twenty-four pleomorphic adenomas and 24 adenoid cystic carcinomas were retrieved. The polymorphism analysis was performed by restriction fragment length polymorphism (RFLP), using the restriction enzymes HphI or AflIII and the mutational screening was performed by PCR-single strand conformational polymorphism (PCR-SSCP). The immunohistochemical analysis was taken by the counting of cells, recorded as the Hscore index, and considering the presence or absence, intensity, distribution and localization of proteins expression. Comparing the two neoplasms, the results demonstrated statistically significant difference for the E-cadherin and β-catenin expression, with pleomorphic adenoma presenting weaker immunostaining. Was observed statistical correlation between E-cadherin and β-catenin expression. CDH1 heterozigotic polymorphism was seen in two cases and 13 cases displayed abnormal mobility electrophoretic shifts, suggesting CTNNB1 gene mutation. The immunohistochemical expression was not statistically correlated with the polymorphism or suggested mutations. In conclusion this study supports that the E-cadherin/β-catenin complex immunohistochemical expression might be related with the myoepithelial component amount and differentiation neither the tumor biological behavior. The cases that showed E-cadherin gene polymorphism presented reduced protein expression and, moreover, CTNNB1 suggested mutations seem not influence in the β-catenin protein expression

Prevalence and antifungal susceptibility of Candida parapsilosis complex isolates collected from oral cavities of HIV-infected individuals

At present, few data are available on the prevalence and antifungal susceptibility of Candida parapsilosis complex isolates from HIV-infected individuals. The C. parapsilosis complex comprises three species, C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. Fifteen of 318 Candida isolates were identified as members of the C. parapsilosis complex by PCR and restriction fragment length polymorphism (RFLP). The prevalence of C. parapsilosis complex isolates was 4.7 %, 2.2 % being identified as C. parapsilosis sensu stricto and 2.5% as C. metapsilosis, while no C. orthopsilosis was isolated. This is believed to be the first study that has identified isolates of C. metapsilosis obtained from the oral cavity of HIV-infected individuals. Antifungal susceptibility tests indicated that all the isolates were susceptible to amphotericin B (AMB), fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC) and caspofungin (CASPO). Although isolates of C. parapsilosis sensu stricto and C. metapsilosis were susceptible to FLC, isolates of C. metapsilosis showed a tendency for higher MICs (>= 1.0 mu g ml(-1)). Based upon the frequency of candidiasis and the fact that certain isolates of the C. parapsilosis complex respond differently to FLC therapy, our data may be of therapeutic relevance with respect to susceptibility and potential resistance to specific antifungal agents. Our data suggest that C. metapsilosis can be a human commensal; its importance as a pathogen has yet to be confirmed.

Genetic characterisation of Giardia duodenalis in dairy cattle in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotypic characterization of Toxoplasma gondii in sheep from Brazilian slaughterhouses: New atypical genotypes and the clonal type II strain identified

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variabilidade genética de Sugarcane mosaic virus, causando mosaico em milho no Brasil

O objetivo deste trabalho foi caracterizar biológica e molecularmente três isolados de Sugarcane mosaic virus (SCMV) de lavouras de milho, analisá-los filogeneticamente e discriminar polimorfismos do genoma. Plantas com sintomas de mosaico e nanismo foram coletadas em lavouras de milho, no Estado de São Paulo e no Município de Rio Verde, GO, e seus extratos foliares foram inoculados em plantas indicadoras e submetidos à análise sorológica com antissoros contra o SCMV, contra o Maize dwarf mosaic virus (MDMV) e contra o Johnsongrass mosaic virus (JGMV). Mudas de sorgo 'Rio' e 'TX 2786' apresentaram sintomas de mosaico após a inoculação dos três isolados, e o DAS-ELISA confirmou a infecção pelo SCMV. O RNA total foi extraído e usado para amplificação por transcriptase reversa seguida de reação em cadeia de polimerase (RT-PCR). Fragmentos específicos foram amplificados, submetidos à análise por polimorfismo de comprimento de fragmento de restrição (RFLP) e sequenciados. Foi possível discriminar os genótipos de SCMV isolados de milho de outros isolados brasileiros do vírus. Alinhamentos múltiplos e análises dos perfis filogenéticos corroboram esses dados e mostram diversidade nas sequências de nucleotídeos que codificam para a proteína capsidial, o que explica o agrupamento separado desses isolados e sugere sua classificação como estirpes distintas, em lugar de simples isolados geográficos.

Genotyping of Brazilian Giardia duodenalis human axenic isolates

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

Carcinogênese de cabeça e pescoço: impacto do polimorfismo MTHFD1 G1958A

OBJETIVO: Investigar o polimorfismo MTHFD1 G1958A envolvido no metabolismo do folato no risco para o câncer de cabeça e pescoço e verificar a associação entre esse polimorfismo com fatores de risco e características clínico-histopatológicas. MÉTODOS: Estudo retrospectivo que avaliou o polimorfismo MTHFD1 G1958A em 694 indivíduos (240 pacientes e 454 controles), por meio da técnica de análise de polimorfismo de comprimento de fragmento de restrição. Para análise estatística foram utilizados os testes de regressão logística múltipla e qui-quadrado. RESULTADOS: Tabagismo e idade superior a 42 anos foram preditores da doença (p < 0,05). Os genótipos MTHFD1 1958GA ou AA foram associados ao tabagismo (p = 0,04) e etilismo (p = 0,03) e estão presentes em maior proporção em tumores com estádios mais avançados (p = 0,04) e em pacientes com menor sobrevida (p = 0,03). CONCLUSÃO: A presença do polimorfismo MTHFD1 G1958A associada aos hábitos tabagista e etilista aumenta o risco para desenvolvimento de câncer de cabeça e pescoço.

Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em. Thell.)

Restriction fragment length polymorphism (RFLP) maps of chromosomes 6A, 6B, and 6D of hexaploid wheat (Triticum aestivum L. em. Thell.) have been produced. They were constructed using a population of F 7-8 recombinant inbred lines derived from a synthetic wheat X bread wheat cross. The maps consist of 74 markers assigned to map positions at a LOD ≥ 3 (29 markers assigned to 6A, 24 to 6B, and 21 to 6D) and 2 markers assigned to 6D ordered at a LOD of 2.7. Another 78 markers were assigned to intervals on the maps. The maps of 6A, 6B, and 6D span 178, 132, and 206 cM, respectively. Twenty-one clones detected orthologous loci in two homoeologues and 3 detected an orthologous locus in each chromosome. Orthologous loci are located at intervals of from 1.5 to 26 cM throughout 70% of the length of the linkage maps. Within this portion of the maps, colinearity (homosequentiality) among the three homoeologues is strongly indicated. The remainder of the linkage maps consists of three segments ranging in length from 47 to 60 cM. Colinearity among these chromosomes and other Triticeae homoeologous group 6 chromosomes is indicated and a consensus RFLP map derived from maps of the homoeologous group 6 chromosomes of hexaploid wheat, tetraploid wheat, Triticum tauschii, and barley is presented.

Toxoplasma gondii genotyping in a dog co-infected with distemper virus and ehrlichiosis rickettsia

This paper reports a toxoplasmosis, erhlichiosis and distemper co-infection in a dog with an exuberant neuropathological clinical picture. Primary involvement was discussed based on information collected in the analysis of the clinical case, such as neurological impairment, epidemiological data, poor immunoprophylactic scheme of the dog affected and the role of these diseases on immunosuppression. Canine distemper and ehrlichiosis were diagnosed based on epidemiologic data, clinical signs, hematological and cytological evaluation. Toxoplasma gondii was isolated and genetically characterized as Type I using restriction analysis (RFLP) with SAG-2 genes. Immunosuppression features of both dogs and human beings are discussed, as well as implications on animal and public health. This is the first report on toxoplasmosis, ehrlichiosis and distemper co-infection in a dog in Brazil, associated with genotyping determination of the T. gondii strain involved.