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Edible bird's nests (EBNs) are the nests of swiftlets, made from the saliva of the male swiftlet (Aerodramus fuciphagus). Due to their nutritional value, EBNs are recognized as a premium food and highly in demand among the Chinese community. EBNs are commonly adulterated with cheaper ingredients and efforts are being made to combat these activities using different analytical techniques. In this study, nuclear magnetic resonance (1H-NMR) metabolomic fingerprinting combined with chemometrics, particularly principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA), was employed to detect adulteration in EBNs. Authentic EBN samples from different locations in Malaysia were used and adulteration was simulated using nutrient agar, collagen, gelatine, karaya gum and melamine at 1, 5 and 10% w/w, respectively. Overall, unsupervised PCA was able to distinguish authentic EBNs from those adulterated with nutrient agar, collagen and gelatine down to 5% w/w adulteration level. As for EBN adulterated with karaya gum and melamine, a distinct peak can be observed at 1.91 ppm and 6.10 ppm, respectively. The supervised OPLS-DA predictive model was able to differentiate authentic EBNs from simulated adulterated EBNs with 100% accuracy. Conclusively, 1H-NMR metabolomics combined with chemometrics could be a potential tool for the detection of adulteration in EBN.

As stingless bee honey (SBH) is gaining in popularity in the Malaysian market, it is now prone to adulteration. The higher price of SBH compared to floral honey has led to the use of unusual adulterants such as vinegar and even floral honey to mimic the unique taste and appearance of SBH. Since the current AOAC 998.12 method fails to detect these adulterants as their δ13C values are in the range for C3 plants, untargeted 1H NMR metabolomics was proposed. Principal component analysis of SBH 1H NMR fingerprints was able to distinguish authentic SBHs from adulterated ones down to 1% adulteration level for selected adulterants. Discriminant analysis showed promising results in distinguishing the preliminary datasets of authentic SBHs from the adulterated ones, including discriminating SBHs adulterated with different adulterants derived from C3 and C4 plants. Hence, to assure any emerging adulterant can be detected, all 1H NMR regions should be considered.

It is important to understand the Amazonian Dark Earth (ADE) diversified microbial communities which colonize agricultural soils and interact with plants, allowing a more sustainable way of soil utilization. Genomic prospection of biotechnological interest, such as the phenazine biosynthesis genes, was carried out by the characterization of the bacterial community structure through the analysis of the 16S rRNA gene in ADE rhizospheric and bulk soil sampled in the forest, and in the agriculture managed soil, being subsequently cultivated with caupi bean. Additionally, the phzF gene coding for a key enzyme in the phenazine biosynthesis was detected and quantified. Gene polymophism (Terminal Restriction Fragment Length Polymorphism, T-RFLP) analysis revealed differences in the bacterial community structure between colonized rhizospheric and bulk soil, but there were no differences concerning the 16S rRNA gene copy number. Besides, the phzF gene copy number was higher in the rhizospheric than in the bulk soil, without any difference between forest and agricultural soils. This work confirms that the type of soil and the interaction between plants and microorganisms are key factors that shape the structure and diversity of bacterial communities and represent a biotechnological potential, with the possibility of finding natural compounds for use in biological control. Key words: Amazonian Dark Earth, rhizosphere, quantitative polymerase chain reaction (PCR), terminal restriction fragment length polymorphism (T-RFLP), microbial diversity.

An untargeted metabolomics approach was applied for the discrimination of honey of various floral and geographical origins using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QToF MS). Evaluation of data by multivariate data analysis allowed the discrimination of New Zealand and Australian honeys from honeys originating in other countries. It was also possible to differentiate between mono- and polyfloral honeys within one region, as well as between some monofloral honeys of various geographical origins. Characteristic markers, contributing to the segregation of different honey types, were selected and characterized.

Amazonian Dark Earth (ADE) is a highly fertile soil of anthropogenic origin characterized by higher amount of charred black carbon (BC). ADE is considered a fertility model, however knowledge about the fungal community structure and diversity inhabiting ADE and BC is scarce. Fungal community structure and diversity of ADE and BC from four sites under different land use (three agricultural systems and a secondary pristine forest) in the Brazilian Central Amazon was evaluated by 18S rRNA gene pyrosequencing. Fungal communities in ADE and BC were dissimilar and showed differential abundances of fungal Operational Taxonomic Units (OTUs). Estimated fungal species richness (ACE and Chao-1) and diversity (Shannon and Simpson's reciprocal) indices were higher in ADE than BC in all agricultural areas. No differences were observed in the same estimators in ADE and BC samples under secondary forest. Pezizomycotina fungi, and OTUs assigned to Cordyceps confragosa, Acremonium vitellinum, Camarops microspora and Hirsutella rhossiliensis were more abundant in BC particles than in ADE. This study is a breakthrough in understanding the fungal communities in BC particles from ADE and can also be valuable in future studies considering biochar application in soil.

Amazon rainforest has been subjected to particularly high rates of deforestation caused mainly by the expansion of cattle pasture and agriculture. A commonly observed response to land-use change is a negative impact on biodiversity of plant and animal species. However, its effect on the soil microbial community and ecosystem functioning is still poorly understood. Here, we used a DNA metagenomic sequencing approach to investigate the impact of land-use change on soil microbial community composition and its potential functions in three land-use systems (primary forest, pasture and secondary forest) in the Amazon region. In general, the microbial community structure was influenced by changes in soil physicochemical properties. Aluminum and water-holding capacity significantly correlated to overall community structure and most of microbial phyla. Taxonomic changes were followed by potential functional changes in the soil microbial community, with pasture presenting the most distinct profile in comparison with other sites. Although taxonomic structure was very distinct among sites, we observed a recovery of the potential functions in secondary forest after pasture abandonment. Our findings elucidate a significant shift in belowground microbial taxonomic and potential functional diversity following natural forest re-establishment and have implications for ecological restoration programs in tropical and sub-tropical ecosystems.

Honey is a high value food commodity with recognized nutraceutical properties. A primary driver of the value of honey is its floral origin. The feasibility of applying multivariate data analysis to various chemical parameters for the discrimination of honeys was explored. This approach was applied to four authentic honeys with different floral origins (rata, kamahi, clover and manuka) obtained from producers in New Zealand. Results from elemental profiling, stable isotope analysis, metabolomics (UPLC-QToF MS), and NIR, FT-IR, and Raman spectroscopic fingerprinting were analyzed. Orthogonal partial least square discriminant analysis (OPLS-DA) was used to determine which technique or combination of techniques provided the best classification and prediction abilities. Good prediction values were achieved using metabolite data (for all four honeys, Q(2)=0.52; for manuka and clover, Q(2)=0.76) and the trace element/isotopic data (for manuka and clover, Q(2)=0.65), while the other chemical parameters showed promise when combined (for manuka and clover, Q(2)=0.43).

The QuEChERS methods, CEN 15662 and AOAC 2007.01, and different clean-up variations were applied to the determination of pesticide residues in Peumus boldus Molina leaves, using LC and GC coupled to single and tandem mass spectrometry. Boldo belongs to the class of medicinal and aromatic plants. Because of its complex chemical composition including terpene peroxides, alkaloids and phenolic acids at concentrations typically 100-fold higher than that of a possible contaminant, it is a very challenging matrix for pesticide residue determination. Thin layer chromatography was used to evaluate the efficacy of removal of matrix co-extractives using different clean-up sorbents, including neutral alumina, graphitized carbon black (GCB), octadecyl reverse phase silica gel (RP-C18) and pH modification in the extraction and clean-up steps. The most promising method was a combination of GCB, RP-C18 and primary-secondary amine (PSA) within the QuEChERS template. The final optimized method was validated according to the SANTE guidelines using LC–MS/MS and GC–MS/MS. The method scope includes 84 representative pesticide residues with recoveries ranging from 70 to 119% and within laboratory relative standard deviations below 15%. The method was initially validated for 25 representative pesticides using GC-MSD, making it applicable in those laboratories equipped only with a single mass detector. It is important to emphasize that the method complies with the requirements of international pharmacopoeias and food regulatory agencies, since herbal teas are found in both categories, as herbal remedies and foods.

Edible bird’s nest (EBN) is widely consumed in South-East Asia as a health food product. In recent years, the adulteration of EBN is quite common due to its high economic value and limited supplies of the natural authentic product. Authentication and traceability of EBN demands time-consuming sequential analytical measurements to confirm that the product quality meets technical and regulatory requirements. Mid Infrared spectroscopy combined with chemometric analysis by Data-Driven Soft Independent Modelling of Class Analogy (DD-SIMCA) was used in this study to screen Malaysian EBNs for structural adulterants and for confirmation of geographical origin. The samples used were authentic processed EBNs of different grades (2A, 3A, 4A and 5A) from the swiftlet species Aerodramus fuciphagus supplied by Ministry of Health, Malaysia. Similarly, adulterants commonly used in EBN namely agar, Karaya gum, bovine gelatin, fish skin, melamine and porcine gelatine were also characterized. Adulterant mixtures were prepared gravimetrically at concentrations of 1, 5 and 10%w/w by combining appropriate quantities of adulterants with individual authentic EBN samples. Samples were then analyzed by Mid-Infrared – Attenuated Total Reflectance (MIR-ATR) spectroscopy. All test samples are properly categorized as members of the target class. However, 61% of the tested adulterated EBNs were correctly classified as ‘alien’ or non-authentic samples. The sensitivity and specifity values for DD-SIMCA models applied to origin determination ranged from 90 to 100% for production regions from Kelantan, Perak and Malacca. MIR-ATR spectroscopy in combination with DD-SIMCA modelling has shown potential as a rapid screening technique to detect the presence adulterants and confirm the provenance of EBN produced in Malaysia. Further work is required to validate the approach with a larger set of authentic samples and for a wider range of adulterants including flavour enhancers and preservatives.

Amazonian Dark Earth (ADE) in the Brazilian Amazon is the main evidence left by pre-Columbian indigenous populations indicating that infertile soils can be transformed into highly fertile ground. Changes in vegetation cover and seasonality are likely to influence microbial communities; however, little is known about these effects on ADE. Therefore, this study compared the effects of two land use systems in ADE and adjacent soil (ADJ) during the rainy and dry seasons using biochemical and molecular tools. Bacterial community function was determined by community level physiological profile (CLPP), bacterial community structure by terminal restriction length polymorphism (T-RFLP), and bacterial community composition by pyrosequencing of the V4 16S rRNA gene region. Our results show that the community structure is highly affected by vegetation, in both, ADE and ADJ soils. Regarding community function, Average Well Color Development (from Biolog substrates) were higher in ADE than ADJ during the rainy season and kept the same pattern of substrate utilization during the dry season and finally, community composition showed to be influenced even at the level of family, mostly by soil type rather than vegetation. Collectively, our study provides insights into processes affecting the bacterial community assemblages in both, ADE and adjacent soils. Key words: Amazonian soils, vegetation type, seasonality, soil bacteria.

Amazonian Dark Earths (ADE) or Terra Preta de Índio formed in the past by pre-Columbian populations are highly sustained fertile soils supported by microbial communities that differ from those extant in adjacent soils. These soils are found in the Amazon region and are considered as a model soil when compared to the surrounding and background soils. The aim of this study was to assess the effects of ADE and its surrounding soil on the rhizosphere bacterial communities of two leguminous plant species that frequently occur in the Amazon region in forest sites (Mimosa debilis) and open areas (Senna alata). Bacterial community structure was evaluated using terminal restriction fragment length polymorphism (T-RFLP) and bacterial community composition by V4 16S rRNA gene region pyrosequencing. T-RFLP analysis showed effect of soil types and plant species on rhizosphere bacterial community structure. Differential abundance of bacterial phyla, such as Acidobacteria, Actinobacteria, Verrucomicrobia, and Firmicutes, revealed that soil type contributes to shape the bacterial communities. Furthermore, bacterial phyla such as Firmicutes and Nitrospira were mostly influenced by plant species. Plant roots influenced several soil chemical properties, especially when plants were grown in ADE. These results showed that differences observed in rhizosphere bacterial community structure and composition can be influenced by plant species and soil fertility due to variation in soil attributes.

The sterile insect technique (SIT) is a biological control tactic that is used as a component of area-wide integrated pest management (AW-IPM) programs. The SIT can only be applied against disease-transmitting mosquitoes when only sterile male mosquitoes are released, and the blood-sucking and potentially disease-transmitting females are eliminated from the production line. For Anopheles arabiensis, a potent vector of malaria, a genetic sexing strain was developed whereby females can be eliminated by treating the eggs or larvae with the insecticide dieldrin. To evaluate the presence of dieldrin residues in male mosquitoes designated for SIT releases, a simple, sensitive, and accurate gas chromatography-electron capture detector (GC-ECD) method was developed. In addition, bioaccumulation and food chain transfer of these residues to fish after feeding with treated mosquitoes was demonstrated. The overall recovery from method validation studies was 77.3 ± 2.2% (mean ± relative standard deviation [RSD]) for the mosquitoes, and 99.1 ± 4.4% (mean ± RSD) for the fish. The average dieldrin concentration found in adult male An. arabiensis was 28.1 ± 2.9 µg/kg (mean ± standard deviation [SD]). A range of 23.9 ± 1.1 µg/kg to 73.9 ± 5.2 µg/kg (mean ± SD) of dieldrin was found in the fish samples. These findings indicate the need to reassess the environmental and health implications of control operations with a SIT component against An. arabiensis that involves using persistent organochlorines in the sexing process.

Here, we compare the fungal community composition and diversity in Amazonian Dark Earth (ADE) and the respective non-anthropogenic origin adjacent (ADJ) soils from four different sites in Brazilian Central Amazon using pyrosequencing of 18S ribosomal RNA (rRNA) gene. Fungal community composition in ADE soils were more similar to each other than their ADJ soils, except for only one site. Phosphorus and aluminum saturation were the main soil chemical factors contributing to ADE and ADJ fungal community dissimilarities. Differences in fungal richness were not observed between ADE and ADJ soil pairs regarding to the most sites. In general, the most dominant subphyla present in the soils were Pezizomycotina, Agaricomycotina, and Mortierellomycotina. The most abundant operational taxonomic units (OTUs) in ADE showed similarities with the entomopathogenic fungus Cordyceps confragosa and the saprobes Fomitopsis pinicola, Acremonium vitellinum, and Mortierellaceae sp., whereas OTUs similar to Aspergillus niger, Lithothelium septemseptatum, Heliocephala gracillis, and Pestalosphaeria sp. were more abundant in ADJ soils. Differences in fungal community composition were associated to soil chemical factors in ADE (P, Ca, Zn, Mg, organic matter, sum of bases, and base saturation) and ADJ (Al, potential acidity, Al saturation, B, and Fe) soils. These results contribute to a deeper view of the fungi communities in ADE and open new perspectives for entomopathogenic fungi studies. Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0703-7) contains supplementary material, which is available to authorized users.

The study of the ecology of soil microbial communities at relevant spatial scales is primordial in the wide Amazon region due to the current land use changes. In this study, the diversity of the Archaea domain (community structure) and ammonia-oxidizing Archaea (richness and community composition) were investigated using molecular biology-based techniques in different land-use systems in western Amazonia, Brazil. Soil samples were collected in two periods with high precipitation (March 2008 and January 2009) from Inceptisols under primary tropical rainforest, secondary forest (5-20 year old), agricultural systems of indigenous people and cattle pasture. Denaturing gradient gel electrophoresis of polymerase chain reaction-amplified DNA (PCR-DGGE) using the 16S rRNA gene as a biomarker showed that archaeal community structures in crops and pasture soils are different from those in primary forest soil, which is more similar to the community structure in secondary forest soil. Sequence analysis of excised DGGE bands indicated the presence of crenarchaeal and euryarchaeal organisms. Based on clone library analysis of the gene coding the subunit of the enzyme ammonia monooxygenase (amoA) of Archaea (306 sequences), the Shannon-Wiener function and Simpson's index showed a greater ammonia-oxidizing archaeal diversity in primary forest soils (H' = 2.1486; D = 0.1366), followed by a lower diversity in soils under pasture (H' = 1.9629; D = 0.1715), crops (H' = 1.4613; D = 0.3309) and secondary forest (H' = 0.8633; D = 0.5405). All cloned inserts were similar to the Crenarchaeota amoA gene clones (identity > 95 %) previously found in soils and sediments and distributed primarily in three major phylogenetic clusters. The findings indicate that agricultural systems of indigenous people and cattle pasture affect the archaeal community structure and diversity of ammonia-oxidizing Archaea in western Amazon soils. O conhecimento acerca da ecologia de comunidades microbianas do solo em relevantes escalas espaciais é primordial na ampla região da Amazônia devido às mudanças de uso da terra. Neste estudo, a diversidade do Domínio Archaea (estrutura da comunidade) e Archaea oxidadora de amônia (riqueza e composição da comunidade) foi investigada usando-se técnicas moleculares em solos sob diferentes sistemas de uso da terra na Amazônia ocidental brasileira. Amostras de solo foram coletadas em dois períodos distintos de elevada intensidade pluviométrica (março de 2008 e janeiro de 2009) em Cambissolos sob floresta primária, floresta secundária (5 a 20 anos de regeneração), sistemas agrícolas de povos indígenas e pastagem. A eletroforese em gel com gradiente desnaturante de produtos amplificados pela reação em cadeia da polimerase (PCR-DGGE) usando o gene 16S rRNA como marcador filogenético indicou que as estruturas de comunidades de Archaea em solos sob cultivo agrícola e pastagem são diferentes daquelas encontradas em solos sob floresta primária. Estas, por sua vez, foram mais similares às estruturas de comunidades em solos sob floresta secundária. A análise de sequências de bandas excisadas de géis de DGGE revelou a presença de organismos dos filos Crenarchaeota e Euryarchaeota. Com base em análise de bibliotecas de clones do gene que codifica para a subunidade A da enzima amônia monoxigenase (amoA ) de Archaea (306 sequências), os índices de Shannon-Wiener e Simpson mostraram maior diversidade de Archaea oxidadora de amônia em solos sob floresta primária (H' = 2,1486; D = 0,1366), seguido por menor diversidade em solos sob pastagem (H' = 1,9629; D = 0,1715), cultivo agrícola (H' = 1,4613; D = 0,3309) e floresta secundária (H' = 0,8633; D = 0,5405). Todos os insertos clonados foram similares a clones do gene amoA de Crenarchaeota (identidade >95 %) previamente detectados em solos e sedimentos; e distribuíram-se em três agrupamentos filogenéticos principais. Os resultados indicaram que a estrutura de comunidades de Archaea e a diversidade de Archaea oxidadora de amônia na Amazônia ocidental são afetadas em solos sob sistemas agrícolas de povos indígenas e pastagem.

Studies on keratinolytic microorganisms have been mainly related to their biotechnological applications and association with animal pathologies. However, these organisms have an ecological relevance to recycling keratinous residues in nature. This work aimed to select and identify new culturable feather-degrading bacteria isolated from soils of Brazilian Amazon forest and Atlantic forest. Bacteria that were isolated from temperate soils and bacteria from Amazonian basin soil were tested for their capability to grow on feather meal agar (FMA). Proteolytic bacteria were tested for feather degradation and were further identified according to their morphological and biochemical characteristics. Also, molecular identification based on 16S rDNA gene sequencing was carried out. A total of 24 proteolytic and 20 feather-degrading isolates were selected; Most of the isolates were from the Bacillus genus (division Firmicutes), but one Aeromonas, two Serratia (γ-Proteobacteria), and one Chryseobacterium (Cytophaga–Flavobacterium group).

The Anthropogenic Amazon Dark Earth soil is considered one of the world's most fertile soils. These soils differs from conventional Amazon soils because its higher organic content concentration. Here we describe the metagenome sequencing of microbial communities of two sites of Anthropogenic Amazon Dark Earth soils from Amazon Rainforest, Brazil. The raw sequence data are stored under Short Read Accession number: PRJNA344917.

The processes of land conversion and agricultural intensification are a significant cause of biodiversity loss, with consequent negative effects both on the environment and the sustainability of food production.The anthrosols associated with pre-Colombian settlements in the Amazonian region are examples of how anthropogenic activities may sustain the native populations against harsh tropical environments for human establishment, even without a previous intentionality of anthropic soil formation. In a case study (Model I—“Slash-and-Burn”) the community structures detected by automated ribosomal intergenic spacer analysis (ARISA) revealed that soil archaeal, bacterial and fungal communities are heterogeneous and each capable of responding differently to environmental characteristics. ARISA data evidenced considerable difference in structure existing between microbial communities in forest and agricultural soils. In a second study (Model II—“Anthropogenic Soil”), the bacterial community structures revealed by terminal restriction fragment length polymorphism (T-RFLP) differed among an Amazonian Dark Earth (ADE), black carbon (BC) and its adjacent non-anthropogenic oxisoil. The bacterial 16S rRNA gene (OTU) richness estimated by pyrosequencing was higher in ADE than BC. The most abundant bacterial phyla in ADE soils and BC were Proteobacteria—24% ADE, 15% BC; Acidobacteria—10% ADE, 21% BC; Actinobacteria—7% ADE, 12% BC; Verrucomicrobia, 8% ADE; 9% BC; Firmicutes—3% ADE, 8% BC. Overall, unclassified bacteria corresponded to 36% ADE, and 26% BC. Regardless of current land uses, our data suggest that soil microbial community structures may be strongly influenced by the historical soil management and that anthrosols in Amazonia, of anthropogenic origins, in addition to their capacity of enhancing crop yields, may also improve microbial diversity, with the support of the black carbon, which may sustain a particular and unique habitat for the microbes.

The Rodrigo de Freitas lagoon (RFL) is a tropical eutrophic coastal ecosystem located in the urban area of Rio de Janeiro, Brazil. This environment consists of freshwater but has communication with the ocean through a channel (Jardim de Alah’s Channel). The aim of this study was to evaluate the influence of lagoon water on the nearby ocean using molecular and traditional microbiological methods. We hypothesised that due to the eutrophic low-salinity environment, the bacterioplankton community from the RFL would have a native “brackish” composition influenced by both freshwater and marine phylotypes, and that bacterial phylotypes of this community would be detected in oceanic samples closer to the channel between the lagoon and the ocean. The cultivation and microscopy experiments clearly showed this influence. Bacterial cell counts revealed that the greater amounts of bacterial cells present in the lagoon increased the observed values seen at oceanic stations near the channel. The Denaturing gradient gel eletrophoresis community profiles also showed a clear influence of Rodrigo de Freitas lagoon waters on the adjacent beaches. The band patterns found for the stations near the channel showed that these communities were mixtures of the communities of the lagoon and sea, and as the distance from the channel increased, the samples became more similar to ocean bacterial communities. A 16S rRNA gene clone library was constructed using a sample acquired from the connection point between the lagoon and the ocean. Around 52% of the sequences in the library showed similarity to the genus Proteobacteria (1% Alpha, 21% Beta, 19% Gamma and 29% unclassified Proteobacteria), and the second most abundant genus was Bacteroidetes, with 15% of the total clones. The results showed that the structure of the bacterial community had both freshwater and marine characteristics.

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml(-1). The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC50 values ranged from 0.3 ng mL(-1) to 5.5 ng mL(-1) by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL(-1) to 1.7 ng mL(-1) by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserum G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using D1). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (G1) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1).

Chloramphenicol is a broad-spectrum antibiotic shown to have specific activity against a wide variety of organisms that are causative agents of several disease conditions in domestic animals. Chloramphenicol has been banned for use in food-producing animals for its serious adverse toxic effects in humans. Due to the harmful effects of chloramphenicol residues livestock products should be free of any traces of these residues. Several analytical methods are available for chloramphenicol analysis but sensitive methods are required in order to ensure that no traces of chloramphenicol residues are present in edible animal products. In order to prevent the illegal use of chloramphenicol, regulatory control of its residues in food of animal origin is essential. A competitive enzyme-linked immunosorbent assay for chloramphenicol has been locally developed and optimized for the detection of chloramphenicol in sheep serum. In the assay, chloramphenicol in the test samples and that in chloramphenicol-horseradish peroxidase conjugate compete for antibodies raised against the drug in camels and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H2O2) is used as chromogen-substrate system. The assay has a detection limit of 0.1 ng/mL of serum with a high specificity for chloramphenicol. Cross-reactivity with florfenicol, thiamphenicol, penicillin, tetracyclines and sulfamethazine was not observed. The assay was able to detect chloramphenicol concentrations in normal sheep serum for at least 1 week after intramuscular injection with the drug at a dose of 25 mg/kg body weight (b.w.). The assay can be used as a screening tool for chloramphenicol use in animals.

As Terras Pretas de Índio (TPI), também conhecida por Amazonian Dark Earth, apresentam horizonte A antrópico, elevado pH, nutrientes importantes para o crescimento das plantas, elevado teor de carvão pirogênico e intensa atividade biológica quando comparadas aos seus solos de origem. A comunidade microbiana do solo é essencial para o funcionamento dos ecossistemas, sendo fundamentais em processos de decomposição da matéria orgânica, na disponibilização de nutrientes para as plantas e na ciclagem de nutrientes. Este estudo teve como objetivo acessar as estruturas e composição das comunidades de Bacteria e Archaea em fragmentos de carvão pirogênico (provenientes de TPI), TPI e solo adjacente (ADJ) utilizando as técnicas moleculares. Os solos foram coletados em quatro sítios arqueológicos (Balbina, Barro Branco, Costa do Açutuba e Hatahara), localizados na Amazônia Central. Em geral, as TPIs apresentaram elevados valores de pH, P e Ca corroborando com resultados anteriores em TPIs. As amostras de TPI dos sítios Balbina, Barro Branco e Hatahara apresentaram maior número de cópias do gene 16S rRNA de Bacteria quando comparadas com as amostras de solo ADJ. Os resultados obtidos pelas técnicas de fingerprinting (Terminal Restriction Fragment Length Polymorphism - T-RFLP e Denaturing Gradient Gel Electrophoresis - DGGE) utilizando o gene 16S rRNA de Bacteria e Archaea revelaram que as estruturas das comunidades bacterianas em fragmentos de carvão apresentaram diferenças significativas quando comparados com os solos ADJ. A partir da técnica de pirosequenciamento, os filos Proteobacteria, Actinobacteria e Crenarchaeota apresentaram predominância nos fragmentos de carvão. Observou-se também que em fragmentos de carvão, os microorganismos encontrados podem estar diretamente relacionados aos ciclos do N e C. Além disso, a presença desses micro-organismos em fragmentos de carvão pode favorecer a microbiota do solo e, consequentemente, sua qualidade. Neste sentido, o carvão pode também servir como elemento de recuperação em ambientes degradados, agindo como condicionador do solo sendo esta uma alternativa promissora no manejo de solos agrícolas. Amazonian Dark Earth (ADE), also known as Terra Preta de Índio, present tropical A horizon, high pH, important nutrients for plant growth, high pyrogenic charcoal levels and intense biological activity when compared to its soil of origin. Soil microbial community is essential for ecosystem function, which is involved in fundamental processes such as the decomposition of organic matter, availability of nutrients to plants and the nutrient cycling. The objective of this study was to assess the structure and composition of bacterial and archaeal communities in fragments of pyrogenic charcoal (from ADE), ADE and adjacent soil (ADJ) using molecular techniques. The soils were collected in four archaeological sites (Balbina, Barro Branco, Costa do Açutuba and Hatahara), located in the central Amazon. In general, ADE presented high values of pH, P and Ca agreeing with previous results in ADE. ADE samples in Balbina, Barro Branco and Hatahara sites presented higher bacterial 16S rRNA gene copy number in comparison with ADJ soil samples. The results obtained with the fingerprinting techniques (Terminal Restriction Length Polymorphism, T-RFLP and Denaturing Gradient Gel Electrophoresis, DGGE) using the bacterial and archaeal 16S rRNA genes revealed that bacterial community structure in charcoal fragments differed significantly when compared to the ADJ soils. Using the pyrosequencing technique, the phyla Proteobacteria, Actinobacteria and Crenarchaeota were predominant in charcoal fragments. It was also observed that microorganisms from charcoal fragments may be directly related to the N and C cycles. Furthermore, the presence of these microorganisms in charcoal fragments may favor the soil microbiota and, consequently, its quality. In this context, pyrogenic charcoal can serve as an element to recover degraded areas acting as soil conditioner and a promising alternative to the management of agricultural soils.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:15:29Z No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Made available in DSpace on 2014-02-26T17:15:29Z (GMT). No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Previous issue date: 2003-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:25:38Z No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Made available in DSpace on 2014-05-20T15:25:38Z (GMT). No. of bitstreams: 1 WOS000180834300035.pdf: 1288581 bytes, checksum: c80b0ab1625eeba1cd15e9da6e465b26 (MD5) Previous issue date: 2003-02-01 Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies. Univ São Paulo, Inst Biociencias, Dept Bot, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Quim, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Ciências Biomed, BR-05508900 São Paulo, Brazil Univ São Paulo, Escola Super Agr Luiz de Queiroz, BR-13418900 Piracicaba, Brazil Univ São Paulo, Ctr Energia Nucl Agr, BR-13400970 Piracicaba, Brazil Inst Agron Campinas, Ctr Citricultura Sylio Moreira, BR-13490970 Cordeiropolis, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet, BR-14884900 Jaboticabal, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Inst Biol, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Inst Comp, BR-13083970 Campinas, SP, Brazil Univ São Paulo, Fac Ciências Farmaceut, BR-14040901 Ribeirao Preto, Brazil Univ São Paulo, Fac Filosofia Ciências & Letras, BR-14040901 Ribeirao Preto, Brazil Inst Ludwig Pesquisa Canc, BR-01509010 São Paulo, Brazil Univ Mogi das Cruzes, Nucleo Integrado Biotecnol, BR-08780911 Mogi das Cruzes, Brazil Inst Agron Campinas, BR-13001970 Campinas, SP, Brazil Inst Biol, BR-04014002 São Paulo, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciências Agron, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, BR-18618000 Botucatu, SP, Brazil Univ Fed Sao Carlos, BR-13600970 São Paulo, Brazil Univ Fed Mato Grosso do Sul, Dept Comp & Estatist, BR-79070900 Campo Grande, MS, Brazil USDA ARS, Parlier, CA 93648 USA Univ Estadual Paulista, Fac Ciências Agr & Vet, BR-14884900 Jaboticabal, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Ciências Agron, BR-18603970 Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, BR-18618000 Botucatu, SP, Brazil

A survey was carried out to investigate the prevalence of nicarbazin residues in eggs in Northern Ireland. Nicarbazin, in the form of 4,4'-dinitrocarbanilide (DNC), was detected in 39 of the 190 eggs analysed. An experiment was designed to establish the relationship between nicarbazin-contaminated feed and nicarbazin residues in eggs. The concentrations of both the DNC and 4,6-dimethyl-2-hydroxypyrimidine (DHP) components of the drug in eggs were proportional to feed levels. The maximum feed nicarbazin concentration of 12.1 mg/kg (8.6 mg/kg DNC and 3.5 mg/kg DHP) gave rise to mean maximum whole egg concentrations of 631 micrograms/kg DNC and 51.8 micrograms/kg DHP. After withdrawal of the experimental diet, DNC was undetectable in eggs after 12 days and DHP after 3 days. Feed contaminated with nicarbazin at concentrations greater than about 2 mg/kg gave rise to egg DNC residues at concentrations greater than the Differential Action Limit (DAL) set by the UK (100 micrograms/kg). DNC was contained almost entirely in the yolk of the egg, whereas DHP was distributed between albumen and yolk in a ratio of approximately 3:1.

Plant uptake of toxins and their translocation to edible plant parts are important processes in the transfer of contaminants into the food chain. Atropine, a highly toxic muscarine receptor antagonist produced by Solanacea species, is found in all plant tissues and can enter the soil and hence be available for uptake by crops. The absorption of atropine and/or its transformation products from soil by wheat (Triticum aestivum var Kronjet) and its distribution to shoots was investigated by growing wheat in soil spiked with unlabeled or (14)C-labeled atropine. Radioactivity attributable to (14)C-atropine and its transformation products was measurable in plants sampled at 15 d after sowing (DAS) and thereafter until the end of experiment. The highest accumulation of (14)C-atropine and/or its transformation products by plants was detected in leaves (between 73 and 90% of the total accumulated) with lower amounts in stems, roots, and seeds (approximately 14%, 9%, and 3%, respectively). (14)C-Atropine and/or its transformation products were detected in soil leachate at 30, 60, and 90 DAS and were strongly adsorbed to soil, with 60% of the applied dose adsorbed at 30 DAS, plateauing at 70% from 60 DAS. Unlabeled atropine was detected in shoots 30 DAS at a concentration of 3.9 ± 0.1 μg kg(-1) (mean ± SD). The observed bioconcentration factor was 2.3 ± 0.04. The results suggest a potential risk of atropine toxicity to consumers.

Summary Zeranol and two Fusarium toxins, α-zearalenol and s-zearalenol, were confirmed by gaschromatography/mass spectrometry (GC/MS) in bovine bile samples referred to this laboratory for analysis. No evidence of zeranol abuse was found on-farm. Given the recent suggestion that zeranol might arise from the metabolism of these Fusarium toxins, and the finding of zeranol in bovine and ovine urine across the EU, it was concluded that the residues had arisen as a result of natural metabolism.

Background: The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. Methods: Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. Results: Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. Conclusion: Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Two experiments were carried out to investigate possible causes of nicarbazin residues in broiler chicken tissues. The first experiment was designed to establish whether feeding nicarbazin as stipulated in the product license can result in 4,4'-dinitrocarbanilide (DNC) tissue residues exceeding the JECFA MRL (200 micrograms/kg). It was shown that the MRL was exceeded in the livers of broilers housed on deep litter, but not in those of broilers housed on wire flooring. Muscle DNC concentrations were well below the MRL. The higher residual tissue concentrations in birds housed on deep litter were attributed to faecal recycling. The second experiment was to establish the relationship between nicarbazin-contaminated withdrawal ration up to the point of slaughter and DNC residues in the tissues of broilers that had not been previously exposed to nicarbazin. Tissue DNC concentrations were found to be proportional to feed concentrations. The housing method caused no significant difference in tissue residues. Meal containing nicarbazin at a concentration of 2.4 mg/kg or greater caused liver DNC residues above the JECFA MRL. Violative residues may, therefore, occur in chickens not exposed to nicarbazin during rearing, but fed withdrawal ration contaminated at 2.4 mg/kg or greater, or in chickens housed on deep litter and fed nicarbazin-medicated meal according to the product license even when the withdrawal ration is nicarbazin-free.

The presence or absence of types I, II and III iodothyronine monodeiodinase enzymes (MDI, MDII and MDIII) and their levels of activity in the skin of goats, which were orally dosed for 60 days with 0, 1.1, 2.2, 4.4, 8.8, 17.5, or 35 mg–1kg liveweight day–1of the anti-thyroid, enzyme-inhibiting drug, propylthiouracil ( PTU ), were determined. Contrary to our earlier report that PTU did not influence skin MDII activity, the currect more thorough investigation (in terms of numbers of observations and the efficiency of the enzyme extraction procedure) indicated that doses of 1.1.to 17.5 mg kg–1liveweight induced a 2 to 3 fold increase (P = 0.01) in MDII activity. However, in three of the four goats treated with 35 mg kg–1group, activity was similar to that of control animals. There were no significant differences between treatments in MDIII activity but there was a trend towards lower levels of activity in the goats dosed with 17.5 and 35 mg kg–1. It is concluded that there is significant MDII and MDIII activity in the skin of goats and that although there is none of the PTU -sensitive MDI enzyme, synthesis of T3 within the skin could nevertheless be modified through increases in MDII activity induced by lower T4 concentrations in the circulation caused by PTU . Changes in pattern of fibre moult induced by treatment with low doses of MD-inhibiting drugs may therefore be achieved through this effect. Since MDII and MDIII enzyme activity may be reduced by high doses of PTU , prolonged treatment with high doses of PTU may have adverse effects on skin tissue.

Zeranol, a semi-synthetic oestrogenic growth promoter, was banned in the EU in 1988. The ability of Member States to police the ban on zeranol has been hampered by suggestions from New Zealand and from this laboratory that zeranol may be formed by the in vivo metabolism of naturally occurring Fusarium spp. toxins. The present study demonstrates that zeranol is formed from alpha-zearalenol and zearalenone in vivo and is detected in bovine bile following the oral administration of these compounds. However, it is not detected following administration of beta-zearalenol. These data suggest that hydrogenation of alpha-zearalenol, probably in the rumen, is responsible for the appearance of zeranol. The present study shows that environmental contamination with Fusarium spp. toxins is widespread in Northern Ireland. Fusarium spp. toxins were present in 32% (n = 422) of all bovine bile samples tested for zeranol during 1995. Zeranol itself was confirmed in 6.6% (n = 28) of the samples. However, the mean alpha-zearalenol and beta-zearalenol concentrations in the bile of zeranol-positive animals were 12 and 9 times higher, respectively, than those in the zeranol-negative animals. The alpha-zearalenol concentration always exceeded the zeranol concentration by at least 5:1. This may, in the future, permit differentiation between zeranol abuse and natural contamination.

Germline mutations in TP53 gene are the main cause of cancer susceptibility in the Li-Fraumeni syndrome (LFS) and its variant, Li-Fraumeni-like syndrome (LFL). It is associated with the development of early-onset cancers, including early onset breast cancer, sarcomas, brain tumors and adrenocortical carcinoma. Breast cancer is the most common tumor found in the LFS/LFL accounting for more than 27% of all tumors described. Recently, the 2010 National Comprehensive Cancer Network guidelines for Hereditary Cancer indicated that TP53 germline mutation testing should be considered for women with breast cancer before age 30 years without a BRCA1/BRCA2 mutation detected. The aim of this study is to identify the role of germline mutations in TP53 gene in women with early onset breast cancer who do not carry a BRCA1 or BRCA2 mutation, regardless of family history. Germline DNA was obtained from peripheral blood, collected after genetic counseling and signature of informed consent. TP53 mutation status was assessed by DNA sequencing of the entire coding region of TP53 gene (exons 2-11). A total of 41 females who developed ductal invasive breast cancer were selected, including (a) 15 women diagnosed with very early breast cancer (VEBC), before age 30, regardless of family history of cancer and (b) 26 women diagnosed with early breast cancer (EBC), between ages 30 and 36 years old, regardless of family history of cancer. One patient from the VEBC was found to be carrier of a germline missense mutation detected occurred in the DNA binding domain of the TP53 gene, p.V143M, (GTG Financial support: FAPESP Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5598. doi:10.1158/1538-7445.AM2011-5598

A sensitive, accurate and reproducible assay for the determination of residual concentrations of ivermectin in the tissues of farmed Atlantic salmon is described. The assay employs the clean-up of an acetonitrile extract of salmon tissue on a Bond-Elut C8 column, followed by derivatization with trifluoroacetic acid anhydride. Following separation using HPLC, 22,23-dihydroavermectin B1a is determined using fluorescence detection. The limit of detection of the assay is 1 ng per g tissue. Liver, taken from fish treated experimentally with ivermectin, was found to contain higher concentrations of 22,23-dihydroavermectin B1a than any other tissue examined. The half-life of 22,23-dihydroavermectin B1a was similar in all tissues studied, ranging from 89 to 98 degree days.

The changes in the activities of the two vitamin B12-dependent enzymes methylmalonyl-CoA mutase (EC5.4.99.2) and methionine synthetase (5-methyltetrahydrofolate–homocysteine methyltransferase,EC2.1.1.13) are described in two groups of sheep maintained for 20 weeks on either a cobalt-deficient or a Co-sufficient whole-barley diet. At the end of that period, the plasma concentrations of vitamin B12were depressed and those of methylmalonic acid were raised in the Co-deficient group. During the course of the experiment hepatic holo-mutase activity, measured on biopsy samples, declined in Co-deficient animals with a half-life of 73 d. There was a similar, but slower decline in lymphocyte holo-mutase activity which fell with a half-life of 125 d. At slaughter, there was no difference between Co-sufficient and Co-deficient animals in total mutase activity in liver, kidney, brain and spinal cord. In contrast, the total-synthetase activity of liver and kidney was reduced by 60 and 30% respectively in the Co-deficient animals. There was no change in either group of animals in total-synthetase activity, or in either holo-mutase or holo-synthetase activity, in brain and spinal cord. In the Co-deficient animals, holo-mutase and holo-synthetase activities in liver, the tissue with the greatest activity of both enzymes, fell to 25 and 39% respectively, of that of Co-sufficient animals. The corresponding reductions for kidney were 12 and 51 % respectively. These results indicated that activity of both holoenzymes is greatly reduced in Co-deficient sheep.