Detecção molecular de Streptococus agalactiae em amostras de leite bovino obtidas de tanques de expansão

Pós-graduação em Medicina Veterinária - FMVZ

Epidemiologia clássica e molecular da tuberculose pulmonar em pacientes da região norte de Minas Gerais

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Genetic Identification of F1 and Post-F1 Serrasalmid Juvenile Hybrids in Brazilian Aquaculture

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

Identificação molecular de espécies da Subfamília Corydoradinae (Siluriformes: Callichthyidae)

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

Molecular analysis of Aspergillus section Flavi isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within beta-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.

Species identification, distributioin and reproductive interactions of common smoot-hound and blackspotted smooth-hound shark (Genus mustelus) in the Adriatic Sea

Longstanding taxonomic ambiguity and uncertainty exist in the identification of the common (M. mustelus) and blackspotted (M. punctulatus) smooth-hound in the Adriatic Sea. The lack of a clear and accurate method of morphological identification, leading to frequent misidentification, prevents the collation of species-specific landings and survey data for these fishes and hampers the delineation of the distribution ranges and stock boundaries of the species. In this context, adequate species-specific conservation and management strategies can not be applied without risks of population declining and local extinction. In this thesis work I investigated the molecular ecology of the two smooth-hound sharks which are abundant in the demersal trawl surveys carried out in the NC Adriatic Sea to monitor and assess the fishery resources. Ecological and evolutionary relationships were assessed by two molecular tests: a DNA barcoding analysis to improve species identification (and consequently the knowledge of their spatial ecology and taxonomy) and a hybridization assay based on the nuclear codominant marker ITS2 to evaluate reproductive interactions (hybridization or gene introgression). The smooth-hound sharks (N=208) were collected during the MEDITS 2008 and 2010 campaigns along the Italian and Croatian coasts of the Adriatic Sea, in the Sicilian Channel and in the Algerian fisheries. Since the identification based on morphological characters is not strongly reliable, I performed a molecular identification of the specimens producing for each one the cytochrome oxidase subunit 1 (COI) gene sequence (ca. 640 bp long) and compared them with reference sequences from different databases (GenBank and BOLD). From these molecular ID data I inferred the distribution of the two target species in the NC Adriatic Sea. In almost the totality of the MEDITS hauls I found no evidence of species sympatry. The data collected during the MEDITS survey showed an almost different distribution of M. mustelus (confined along the Italian coasts) and M. punctulatus (confined along the Croatian coasts); just one sample (Gulf of Venice, where probably the ranges of the species overlap) was found to have catches of both the species. Despite these data results suggested no interaction occurred between my two target species at least during the summertime (the period in which MEDITS survey is carried out), I still wanted to know if there were inter-species reproductive interactions so I developed a simple molecular genetic method to detect hybridization. This method is based on DNA sequence polymorphism among species in the nuclear ribosomal Internal Transcribed Spacer 2 locus (ITS2). Its application to the 208 specimens collected raised important questions regarding the ecology of this two species in the Adriatic Sea. In fact results showed signs of hybridization and/or gene introgression in two sharks collected during the trawl survey of 2008 and one collected during the 2010 one along the Italian and Croatian coasts. In the case that it will be confirmed the hybrid nature of these individuals, a spatiotemporal overlapping of the mating behaviour and ecology must occur. At the spatial level, the northern part of the Adriatic Sea (an area where the two species occur with high frequency of immature individuals) could likely play the role of a common nursery area for both species.

New molecular detection tools adapted to emerging rhinoviruses and enteroviruses

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

MQN-Mapplet: Visualization of Chemical Space with Interactive Maps of DrugBank, ChEMBL, PubChem, GDB-11, and GDB-13

The MQN-mapplet is a Java application giving access to the structure of small molecules in large databases via color-coded maps of their chemical space. These maps are projections from a 42-dimensional property space defined by 42 integer value descriptors called molecular quantum numbers (MQN), which count different categories of atoms, bonds, polar groups, and topological features and categorize molecules by size, rigidity, and polarity. Despite its simplicity, MQN-space is relevant to biological activities. The MQN-mapplet allows localization of any molecule on the color-coded images, visualization of the molecules, and identification of analogs as neighbors on the MQN-map or in the original 42-dimensional MQN-space. No query molecule is necessary to start the exploration, which may be particularly attractive for nonchemists. To our knowledge, this type of interactive exploration tool is unprecedented for very large databases such as PubChem and GDB-13 (almost one billion molecules). The application is freely available for download at www.gdb.unibe.ch.

Imported diphyllobothriasis in Switzerland: molecular methods to define a clinical case of Diphyllobothrium infection as Diphyllobothrium dendriticum, August 2010

Following a first clinical case of infection by Diphyllobothrium dendriticum in Switzerland in 2006, we report a second case in the country. The species was identified by molecular methods. In the Swiss, French and Italian subalpine regions, human diphyllobothriasis has seen a comeback since the late 1980's, and Diphyllobothrium latum is usually considered the causative agent of the disease. In addition, several locally acquired and imported clinical infections due to allochthonous Diphyllobothrium species have been documented in the last years. Due to the colonisation potential of these parasites and their probably underestimated presence in the human population, there is a need for discriminating them at the medical laboratory level. Because the morphological characters are very similar among the different taxa, a correct identification requires the use of molecular methods. Molecular identification would improve diagnosis and help monitor the distribution of Diphyllobothrium species in Europe.

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log10 cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.

Development of a standardized vehicle identification number. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

The extent of use of Social Security numbers.

"OAI-06-88-00800."

Development of a simple and fast “DNA extraction kit” for sea food identification and marine species

Seafood products fraud, the misrepresentation of them, have been discovered all around the world in different forms as false labeling, species substitution, short-weighting or over glazing in order to hide the correct identity, origin or weight of the seafood products. Due to the value of seafood products such as canned tuna, swordfish or grouper, these species are the subject of the commercial fraud is mainly there placement of valuable species with other little or no value species. A similar situation occurs with the shelled shrimp or shellfish that are reduced into pieces for the commercialization. Food fraud by species substitution is an emerging risk given the increasingly global food supply chain and the potential food safety issues. Economic food fraud is committed when food is deliberately placed on the market, for financial gain deceiving consumers (Woolfe, M. & Primrose, S. 2004). As a result of the increased demand and the globalization of the seafood supply, more fish species are encountered in the market. In this scenary, it becomes essential to unequivocally identify the species. The traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa, amplified when fish, crustacean or shellfish are commercially transformed. Many fish species show a similar texture, thus the certification of fish products is particularly important when fishes have undergone procedures which affect the overall anatomical structure, such as heading, slicing or filleting (Marko et al., 2004). The absence of morphological traits, a main characteristic usually used to identify animal species, represents a challenge and molecular identification methods are required. Among them, DNA-based methods are more frequently employed for food authentication (Lockley & Bardsley, 2000). In addition to food authentication and traceability, studies of taxonomy, population and conservation genetics as well as analysis of dietary habits and prey selection, also rely on genetic analyses including the DNA barcoding technology (Arroyave & Stiassny, 2014; Galimberti et al., 2013; Mafra, Ferreira, & Oliveira, 2008; Nicolé et al., 2012; Rasmussen & Morrissey, 2008), consisting in PCR amplification and sequencing of a COI mitochondrial gene specific region. The system proposed by P. Hebert et al. (2003) locates inside the mitochondrial COI gene (cytochrome oxidase subunit I) the bioidentification system useful in taxonomic identification of species (Lo Brutto et al., 2007). The COI region, used for genetic identification - DNA barcode - is short enough to allow, with the current technology, to decode sequence (the pairs of nucleotide bases) in a single step. Despite, this region only represents a tiny fraction of the mitochondrial DNA content in each cell, the COI region has sufficient variability to distinguish the majority of species among them (Biondo et al. 2016). This technique has been already employed to address the demand of assessing the actual identity and/or provenance of marketed products, as well as to unmask mislabelling and fraudulent substitutions, difficult to detect especially in manufactured seafood (Barbuto et al., 2010; Galimberti et al., 2013; Filonzi, Chiesa, Vaghi, & Nonnis Marzano, 2010). Nowadays,the research concerns the use of genetic markers to identify not only the species and/or varieties of fish, but also to identify molecular characters able to trace the origin and to provide an effective control tool forproducers and consumers as a supply chain in agreementwith local regulations.

First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens)

BACKGROUND Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals. CASE PRESENTATION In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L'Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa. CONCLUSION To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal.

Genetic-morphometric variation in Culex quinquefasciatus from Brazil and La Plata, Argentina

Variation among natural populations of Culex (Culex) quinquefasciatus Say is associated with different vectorial capacities. The species Cx. quinquefasciatus is present in the equatorial, tropical and subtropical zones in the Brazilian territory, with intermediate forms between Cx. quinquefasciatus and Culex pipiens occurring in regions of latitudes around 33°-35°S. Herein, we studied geographically distinct populations of Cx. quinquefasciatus by genetic characterization and analysis of intra-specific wing morphometrics. After morphological analysis, molecular characterization of Cx. quinquefasciatus and intermediate forms was performed by polymerase chain reaction of the polymorphic nuclear region of the second intron of the acetylcholinesterase locus. Additionally, the morphology of adult female wings collected from six locations was analyzed. Wing centroid sizes were significantly different between some geographical pairs. Mean values of R2/R2+3 differed significantly after pairwise comparisons. The overall wing shape represented by morphometric characters could be divided into two main groupings. Our data suggest that Brazilian samples are morphologically and genetically distinct from the Argentinean samples and also indicated a morphological distinction between northern and southern populations of Brazilian Cx. quinquefasciatus. We suggest that wing morphology may be used for preliminary assessment of population structure of Cx. quinquefasciatusin Brazil