A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Electrospray ionization mass spectrometry screening of piperidine alkaloids from Senna spectabilis (Fabaceae) extracts: Fast identification of new constituents and co-metabolites

The fragmentation pattern of a homologous series of piperidine alkaloids isolated from S. spectabilis was investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). The ESI-MS and ESI-MS/MS analyses of EtOH extracts and fractions from flowers and fruits of S. spectabilis allowed to elucidate the structures of four new compounds. The identification of these co-metabolites, based on the fragmentation patterns of previously isolated compounds, and further confirmed by accurate mass spectrometry defines this technique as a powerful tool to determine the metabolomic profile of species which has pharmacological importance. ©2005 Sociedade Brasileira de Química.

Bionomics and identification of Acromyrmex (Hymenoptera: Formicidae) through an illustrated key

This study is based on sampled data obtained in 60 equally distant municipalities of São Paulo State. In this study, we proposed a tool to identify Acromyrmex species in a practical and agile manner through the use of an illustrated key that demonstrates the morphological attributes of workers, associated with the type of cut plant material, external and internal appearance of nests and nidification sites. Through sampling, eleven species and six subspecies of Acromyrmex occurring in the State were identified, including the first registers of A. diasi and A. subterraneus molestans.

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy. © 2007 Entomological Society of America.

New species of Haplometroides (Digenea: Plagiorchiidae) from Phalotris nasutus (Gomes, 1915) (Serpentes, Colubridae)

A new species of Haplometroides (Digenea, Plagiorchiidae) is described from a specimen of Phalotris nasutus (Gomes, 1915) (Serpentes, Colubridae). The host snake was obtained in the municipality of Corumbá, Mato Grosso do Sul State, Brazil. Trematodes were recovered from esophagus, stomach, and small intestine of the host. The main characteristic of the new species is the vitellaria, which is intercecal, cecal, and extracecal in the preacetabular region. A key for identification of the species in Haplometroides is proposed. © American Society of Parasitologists 2007.

Taenia solium and Taenia saginata: Identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies. © 2007 Elsevier Inc. All rights reserved.

A new cunaxid genus with descriptions of two new species from Brazil (Acari: Prostigmata: Bdelloidea: Cunaxidae)

The genus Pseudobonzia Smiley 1975 is revised and divided into two genera. The genus Pseudobonzia Smiley, 1975, with its type species and five other related species, is retained. The remainder of the group is now known as Coleobonzia gen. nov. of which the main characteristics are provided. Coleobonzia argillae (Den Heyer, 1977) is designated as type species for the new genus. Two species from Brazil, viz. Coleobonzia clava sp. nov. and C. moraesi sp. nov. are described and figured. A key to the two genera as well as a key to the Brazilian and South African species of the new genus are provided. Copyright © 2008 Magnolia Press.

Panoramic view of the occurrence of Yersinia species other than Y. pestis in Brazil

Data on the occurrence of Yersinia species, other than Y. pestis in Brazil are presented. Over the past 40 years, 767 Yersinia strains have been identified and typed by the National Reference Center on Yersinia spp. other than Y. pestis, using the classical biochemical tests for species characterization. The strains were further classified into biotypes, serotypes and phagetypes when pertinent. These tests led to the identification of Yersinia cultures belonging to the species Y. enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii and Y. kristensenii. Six isolates could not be classified in any of the known Yersinia species and for this reason were defined as Non-typable (NT). The bio-sero-phagetypes of these strains were diverse. The following species of Yersinia were not identified among the Brazilian strains by the classical phenotypic or biochemical tests: Y. aldovae, Y. rhodei, Y. mollaretti, Y. bercovieri and Y. ruckeri. The Yersinia strains were isolated from clinical material taken from sick and/or healthy humans and animals, from various types of food and from the environment, by investigators of various Institutions localized in different cities and regions of Brazil.

Revision of the genus Physoclypeus Hendel, 1907 (Diptera, Lauxaniidae), with description of seven new species

The genus Physoclypeus Hendel, 1907 has its distribution restricted to the Neotropical region. In this study, its species have been redescribed, three new combinations have been proposed, three lectotypes have been designated, seven new species have been described, and an identification key to the species is presented. An updated list of species of Physoclypeus is presented as: P. annulatus Hendel, 1925; P. coquilletti (Hendel, 1908); P. farinosus (Hendel, 1925); P. flavus (Wiedemann, 1830); P. hendeli sp. nov. (Type locality, Jamaica, N. Irish Town); P. lineatus (Williston, 1896) new comb.; P. montanus (Becker, 1919) new comb.; P. plaumanni sp. nov. (Type locality, Brazil, Santa Catarina); P. risaraldensis sp. nov. (Type locality, Colombia, Risaralda); P. saltensis sp. nov. (Type locality, Argentina, Salta); P. scutellatus (Curran, 1926) new comb.; P. unimaculatus sp. nov. (Type locality, Mexico, Vera Cruz); P. vitattus sp. nov. (Type locality, Brazil, Santa Catarina) and P. zebrinus sp. nov. (Type locality, Costa Rica, Limón).

Multiplex PCR use for Staphylococcus aureus identification and oxacillin and mupirocin resistance evaluation

Oxacillin-resistant Staphylococcus aureus represents a serious problem in hospitals worldwide, increasing infected patients' mortality and morbidity and raising treatment costs and internment time. In this study, the results of using the Multiplex PCR technique to amplify fragments of the genes femA (specific-species), mecA (oxacillin resistance) and ileS-2 (mupirocin resistance) were compared with those of tests conventionally used to identify S. aureus isolates and ascertain their resistance to drugs. Fifty S. aureus strains were isolated from patients receiving treatment at UNOESTE University Hospital in Presidente Prudente, SP, Brazil. The 686 bp fragment corresponding to the gene femA was amplified and detected in all the isolates. On the other hand, the 310 bp fragment corresponding to the mecA gene was amplified in 29 (58%) of the isolates. All of the isolates showed sensitivity to mupirocin in the agar diffusion test, which was corroborated by the lack of any amplicon of the 456 bp fragment corresponding to the ileS-2 gene, in the PCR bands. The conventional tests to identify S. aureus and detect resistance to oxacillin and mupirocin showed 100% agreement with the PCR Multiplex results. The use of techniques for rapid and accurate identification of bacteria and assessment of their resistance may be valuable in the control of infection by resistant strains, allowing the rapid isolation and treatment of an infected patient. However, the results demonstrate that traditional phenotypic tests are also reliable, though they take more time.

Tracking the fin trade: Genetic stock identification in western Atlantic scalloped hammerhead sharks Sphyrna lewini

Location or stock-specific landing data are necessary to improve management of shark stocks, especially those imperiled by overexploitation as a result of the international shark fin trade. In the current absence of catch monitoring directly at extraction sites, genetic stock identification of fins collected from major market supply chain endpoints offers an overlooked but potentially useful approach for tracing the fins back to their geographical, or stock of, origin. To demonstrate the feasibility of this approach, we used mitochondrial control region (mtCR) sequences to trace the broad geographical origin of 62 Hong Kong market-derived Sphyrna lewini fins. Of these fins 21% were derived from the western Atlantic, where this species is listed as 'Endangered' by the International Union for the Conservation of Nature (IUCN). We also show that S. lewini mtCR sequences are geographically segregated in the western Atlantic (overall ΦST = 0.74, n = 177 sharks), indicating that breeding females either remain close to, or home back to, their natal region for parturition. Mixed stock analysis simulations showed that it is possible to estimate the relative contributions of these mitochondrial stocks to fin mixtures in globally sourced trade hubs. These findings underscore the feasibility of using genetic stock identification to source market-derived shark fins to obtain essential and otherwise unavailable data on exploitation levels, and thus to productively inform stock assessment and management of S. lewini and potentially also of other fished shark species. © Inter-Research 2009.

Isolation and identification of black yeasts by enrichment on atmospheres of monoaromatic hydrocarbons

Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species. © 2010 The Author(s).

Identification of sperm head subpopulations with defined pleiomorphic characteristics in ejaculates of captive Goeldi's monkeys (Callimico goeldii)

The aim of this study was to evaluate the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates from the vulnerable Goeldi's monkey (Callimico goeldii) by using a combination of computerized analysis system and Principal Component Analysis (PCA) methods Each sperm head was measured for four primary spermatozoal head dimensional parameters (area [A (μm2)], perimeter [P (μm)], length [L (μm)] and width [W (μm)]) and three head shape derived parameters (ellipticity [(L/W)], elongation [(L-W)/(L+W)] and rugosity [(4πA/P2)]) Six separate subpopulations (SPs) were identified: SP1, constituted by very large, narrow and very elliptical spermatozoa (A=16.85±1.56μm2, W=2.75±0.42μm and ellipticity=2.16±0.24); SP2, characterized by average sized, short, wide and round spermatozoa (A=15.00±1.92μm2, L=5.06±0.49μm, W=3.51±0.31μm and ellipticity=1.44±0.15); SP3, represented by small, wide and slightly round spermatozoa (A=14.95±1.75μm2, W=3.47±0.29μm and ellipticity=1.48±0.14); SP4 included very small, short and very round spermatozoa (A=14.15±2.38μm2, L=4.90±0.57μm and elongation=0.18±0.05); SP5 consisted of average sized and slightly elliptical spermatozoa (A=15.14±1.72μm2 and ellipticity=1.49±0.14); and SP6 included large and round spermatozoa (A=16.30±1.62μm2 and elongation=0.19±0.04) There were differences in the sperm subpopulation distribution (P<0.001) among the five donors analyzed In conclusion, the results of the current study confirmed that the use of computer sperm analysis methods combined with PCA cluster analyses are useful methods to identify, classify, and characterize different sperm head morphometric subpopulations in neotropical primates Broadening our knowledge of C goeldii sperm morphometric abnormalities as well as developing reliable techniques for sperm evaluation may be essential for ex situ conservation of this threatened species © 2012 Elsevier B.V.

Molecular and morphological data support the existence of a sexual cycle in species of the genus Paracoccidioides

The genus Paracoccidioides includes the thermodimorphic species Paracoccidioides brasiliensis and P. lutzii, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed with Paracoccidioides species, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genus Paracoccidioides, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi and Saccharomyces cerevisiae demonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of both Paracoccidioides species was also detected by realtime PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types. In vitro crossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (MAT1-1) and the high-mobility-group (HMG) domain (MAT1-2), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genus Paracoccidioides. © 2013, American Society for Microbiology.

Characterization of resveratrol content in ten wild species of section Arachis, genus Arachis

Peanut is one of the few plants that synthesizes resveratrol, a phenolic compound of the stilbene class, which has been associated with reduced risk of developing chronic diseases, such as cancer, cardiovascular diseases, skin diseases, pulmonary diseases, diabetes and neurological diseases. Resveratrol was detected in different parts of the peanut plant, including roots, leaves, seeds and their derivatives. The wild species of the Arachis section are also strong candidates to synthesize resveratrol because they are phylogenetically closely related to cultivated peanut. Our objective was to characterize the resveratrol content in ten wild species of Arachis with three different genomes (A, B and K). The plant material was composed of leaves of the ten species treated (test) and not treated (control) with ultraviolet (UV) radiation. The test and control samples were extracted and the identification and quantification of resveratrol was performed using high performance liquid chromatography (HPLC). All species studied synthesized resveratrol and the concentrations ranged from 299.5 μg/g in A. kempff-mercadoi to 819.9 μg/g in A. cardenasii. DPPH antioxidant activity varied between 18.7 % for A. duranensis and 48.2 % in A. simpsonii. The results showed that wild Arachis species are a potential source of alleles for improvement of cultivated peanut, with the aim of achieving higher resveratrol content in leaves. © 2013 Springer Science+Business Media Dordrecht.