Bacterial communities in artisanal raw bovine milk cheeses from the southern region of Brazil / Comunidades bacterianas em queijos artesanais de leite bovino cru da região sul do Brasil

The production of artisanal cheeses made with raw bovine milk has grown in the southern region of Brazil. It is important to obtain information about the risks of this practice, especially concerning food safety. In this study, next-generation sequencing was used to identify and characterize the bacterial communities of artisanal raw milk cheeses. We analyzed one pool of five raw milk samples (control group M1) from different dairy farms and nine pools (M2-M10) of 45 artisanal raw milk cheeses.The characterization of the bacterial communities included 199 species distributed across 59 different genera dispersed among the samples. Among the genera observed, 11 were classified as beneficial to the aroma, flavour, colour, and texture of the cheese. Thirty-one genera were classified as harmful to these characteristics. Another 17 were classified as potential pathogens for animals and humans, including Aeromonas, Bacillus, Cronobacter, Salmonella, Staphylococcus, and bacteria of the coliform group, including E. coli and Klebsiella. There was a significant difference (P < 0.05) in the number of bacterial communities identified between the control group (M1) and the two pools of artisanal raw milk cheeses (M2 and M8). This study demonstrated that next-generation sequencing provides in-depth information on the composition of the microbiota in artisanal raw milk cheeses, characterizing bacterial communities, identifying the wide microbial diversity, and identifying microbial benefits and risks.

Devido ao aumento da produção de queijos artesanais com leite bovino cru na região sul do Brasil, é importante obter informações sobre os riscos desta prática, principalmente no que se refere à segurança do alimento. Neste estudo foi utilizada a técnica de Next Generation Sequencing (NGS) para identificar e caracterizar comunidades bacterianas de queijos artesanais de leite cru. Foram analisados um pool de cinco amostras de leite cru como grupo controle (M1) de diferentes propriedades leiteiras localizadas na região norte do estado do Rio Grande do Sul, e nove pools (M2-M10) de 45 queijos artesanais de leite cru. A caracterização das comunidades bacterianas incluiu 199 espécies distribuídas em 59 gêneros diferentes dispersos entre as amostras. Dentre os gêneros observados, 11 foram classificados como benéficos ao aroma, sabor, cor e textura do queijo, enquanto 31 gêneros foram classificados como prejudiciais a essas características. Outros 17 foram classificados como potenciais patogênicos para animais e humanos, incluindo Aeromonas, Bacillus, Cronobacter, Salmonella, Staphylococcus, bactérias do grupo coliforme como Escherichia coli e Klebsiella. Houve diferença significativa (P < 0,05) entre o número de comunidades bacterianas identificadas no grupo controle (M1) e dois pools de queijos artesanais de leite cru (M2 e M8). Este estudo demonstra que o NGS fornece informações detalhadas sobre a composição da microbiota em queijos artesanais de leite cru, caracterizando comunidades bacterianas, identificando a ampla diversidade microbiana, os benefícios e riscos microbianos.

Embryo biopsies for genomic selection in tropical dairy cattle

Genomic selection has transformed the livestock industry, enabling early-life selection of animals. Biopsy sampling of pre-implantation embryos has been described since 1968. However, it was only after 2010, with the advancement of molecular biology techniques such as whole genomic amplification and SNP Chips, that next-generation sequencing became commercially available for bovine embryos. It is now possible to make decisions about which embryos to transfer not only based on recipients' availability or embryo morphology but also on genomic estimates. This technology can be implemented for a wide spectrum of applications in livestock. In this review, we discuss the use of embryo biopsy for genomic selection and share our experience with Gir and Girolando Brazilian breeding programs, as well as future goals for implementing it in Brazilian bovine in vitro embryo production practices.(AU)

Toxinology in the proteomics era: a review on arachnid venom proteomics

Abstract The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000s. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms (scorpion venom) AND (proteome) for scorpion venomics, and (spider venom) AND (proteome) for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.

Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

Characterization of the seminal bacterial microbiome of healthy, fertile stallions using next-generation sequencing

Abstract High-throughput sequencing studies have shown the important role microbial communities play in the male reproductive tract, indicating differences in the semen microbial composition between fertile and infertile males. Most of these studies were made on human beings but little is known regarding domestic animals. Seminal bacteria studies made in stallions mostly focus on pathogenic bacteria and on their impact on reproductive technology. However, little is known about stallion commensal seminal microflora. That ultimately hinders our capacity to associate specific bacteria to conditions or seminal quality. Therefore, the aim of this study was to characterize the seminal microbial composition of 12 healthy, fertile stallion using next-generation sequencing. Hypervariable region V3 was chosen for bacterial identification. A total of nine phyla was detected. The most abundant ones were Bacteroidetes (46.50%), Firmicutes (29.92%) and Actinobacteria (13.58%). At family level, we found 69 bacterial families, but only nine are common in all samples. Porphyromonadaceae (33.18%), Peptoniphilaceae (14.09%), Corynebacteriaceae (11.32%) and Prevotellaceae (9.05%) were the most representative ones, while the Firmicutes phylum displayed the highest number of families (23, a third of the total). Samples showed high inter-subject variability. Findings previously described in other species notably differ from our findings. Families found in human such as Lactobacillaceae, Staphylococcaceae and Streptococcaceae only represented a 0.00%, 0.17% and 0.22% abundance in our samples, respectively. In conclusion, Porphyromonadaceae, Prevotellaceae, Peptoniphilaceae and Corynebacteriaceae families are highly represented in the seminal microbiome of healthy, fertile stallions. A high variation among individuals is also observed.

Effects of dietary lysozyme on immune response and fecal microflora in both sows and their offspring

Two studies were conducted to investigate the effects of dietary lysozyme on immune response, fecal microflora in sows and their offspring fed lysozyme from late gestation to the onset of lactation, and growth performance in weaned piglets. Four antibiotic-based treatments (chlortetracycline, colistin, and lysozyme) were applied in experiment 1. Lysozyme addition significantly increased final body weight, average daily gain, and average daily feed intake, improved feed:gain ratio (F:G), and decreased diarrhea rate in weaned piglets. In experiment 2, postpartum sows were fed diets either with amoxicillin and cephalosporin (SC) or lysozyme (SE). Piglets from SC sows were administered enrofloxacin and those from SE sows were administered lysozyme. Lysozyme treatment decreased serum IL-1, IL-6, and IL-10, but did not influence IL-8, TNF-α, or IFN-γ in weaned piglets. Sequencing revealed that lysozyme significantly decreased Chao-1 index in sows and weaned piglets, increased Bifidobacterium longum in sows, and Lactobacillus coleohominis, L. mucosae, L. amylovorus, and L. hamsteri in weaned piglets. The results suggest that dietary supplementation of lysozyme improved the growth performance of weaned piglets, and dietary supplementation of lysozyme for sows increased immune function and modulated the intestinal flora structure in sows and their offspring.

Characterization of the seminal bacterial microbiome of healthy, fertile stallions using next-generation sequencing

High-throughput sequencing studies have shown the important role microbial communities play in the male reproductive tract, indicating differences in the semen microbial composition between fertile and infertile males. Most of these studies were made on human beings but little is known regarding domestic animals. Seminal bacteria studies made in stallions mostly focus on pathogenic bacteria and on their impact on reproductive technology. However, little is known about stallion commensal seminal microflora. That ultimately hinders our capacity to associate specific bacteria to conditions or seminal quality. Therefore, the aim of this study was to characterize the seminal microbial composition of 12 healthy, fertile stallion using next-generation sequencing. Hypervariable region V3 was chosen for bacterial identification. A total of nine phyla was detected. The most abundant ones were Bacteroidetes (46.50%), Firmicutes (29.92%) and Actinobacteria (13.58%). At family level, we found 69 bacterial families, but only nine are common in all samples. Porphyromonadaceae (33.18%), Peptoniphilaceae (14.09%), Corynebacteriaceae (11.32%) and Prevotellaceae (9.05%) were the most representative ones, while the Firmicutes phylum displayed the highest number of families (23, a third of the total). Samples showed high inter-subject variability. Findings previously described in other species notably differ from our findings. Families found in human such as Lactobacillaceae, Staphylococcaceae and Streptococcaceae only represented a 0.00%, 0.17% and 0.22% abundance in our samples, respectively. In conclusion, Porphyromonadaceae, Prevotellaceae, Peptoniphilaceae and Corynebacteriaceae families are highly represented in the seminal microbiome of healthy, fertile stallions. A high variation among individuals is also observed.(AU)

Development of microsatellite loci and population genetics of the catfish Pimelodus yuma (Siluriformes: Pimelodidae)

Pimelodus yuma (formerly Pimelodus blochii) is a freshwater fish, endemic to the Colombian Magdalena-Cauca and Caribbean basins that experiences habitat disturbances resulting from anthropogenic activities. Due to the lack of information about the population genetics of this species, this study developed 14 species-specific microsatellite loci to assess the genetic diversity and population structure of samples from the lower section of the Cauca River. The studied species showed genetic diversity levels higher than the average values reported for Neotropical Siluriformes and significant inbreeding levels as was described for some congeners. Furthermore, P. yuma comprises two coexisting genetic groups that exhibit gene flow along the lower section of the Cauca River. This information constitutes a baseline for future monitoring of the genetic diversity and population structure in an anthropic influenced sector of the Magdalena-Cauca basin.(AU)

Pimelodus yuma (anteriormente Pimelodus blochii) es un pez dulceacuícola endémico de las cuencas colombianas Magdalena-Cauca y Caribe que experimenta alteraciones del hábitat como resultado de actividades antropogénicas. Debido a la falta de información sobre la genética poblacional de esta especie, este estudio desarrolló 14 loci microsatélites especie-específicos para evaluar la diversidad genética y la estructura poblacional de muestras de la sección baja del río Cauca. La especie estudiada mostró niveles de diversidad genética más altos que los valores promedio reportados para Siluriformes neotropicales y niveles de endogamia significativos como se describió para algunos congéneres. Además, P. yuma comprende dos grupos genéticos coexistentes que exhiben flujo de genes a lo largo de la sección baja del río Cauca. Esta información constituye una línea base para futuros monitoreos de la diversidad genética y la estructura poblacional en un sector de influencia antrópica de la cuenca Magdalena-Cauca.(AU)

Development of microsatellite loci and population genetics of the catfish Pimelodus yuma (Siluriformes: Pimelodidae)

Pimelodus yuma (formerly Pimelodus blochii) is a freshwater fish, endemic to the Colombian Magdalena-Cauca and Caribbean basins that experiences habitat disturbances resulting from anthropogenic activities. Due to the lack of information about the population genetics of this species, this study developed 14 species-specific microsatellite loci to assess the genetic diversity and population structure of samples from the lower section of the Cauca River. The studied species showed genetic diversity levels higher than the average values reported for Neotropical Siluriformes and significant inbreeding levels as was described for some congeners. Furthermore, P. yuma comprises two coexisting genetic groups that exhibit gene flow along the lower section of the Cauca River. This information constitutes a baseline for future monitoring of the genetic diversity and population structure in an anthropic influenced sector of the Magdalena-Cauca basin.(AU)

Pimelodus yuma (anteriormente Pimelodus blochii) es un pez dulceacuícola endémico de las cuencas colombianas Magdalena-Cauca y Caribe que experimenta alteraciones del hábitat como resultado de actividades antropogénicas. Debido a la falta de información sobre la genética poblacional de esta especie, este estudio desarrolló 14 loci microsatélites especie-específicos para evaluar la diversidad genética y la estructura poblacional de muestras de la sección baja del río Cauca. La especie estudiada mostró niveles de diversidad genética más altos que los valores promedio reportados para Siluriformes neotropicales y niveles de endogamia significativos como se describió para algunos congéneres. Además, P. yuma comprende dos grupos genéticos coexistentes que exhiben flujo de genes a lo largo de la sección baja del río Cauca. Esta información constituye una línea base para futuros monitoreos de la diversidad genética y la estructura poblacional en un sector de influencia antrópica de la cuenca Magdalena-Cauca.(AU)

Population genetics of the endangered catfish Pseudoplatystoma magdaleniatum (Siluriformes: Pimelodidae) based on species-specific microsatellite loci

The Neotropical catfish genus Pseudoplatystoma comprises eight species of large size, widely distributed in South American basins. The endangered species P. magdaleniatum is endemic to Magdalena basin (Colombia), experiences high fishing pressure and its population genetics is relatively unknown. To study the genetic status and structure of P. magdaleniatum, 25 species-specific polymorphic microsatellite loci were developed using next-generation sequencing and then tested in samples collected in the Magdalena-Cauca basin. Based on 15 of these loci, P. magdaleniatum showed a high number of alleles per locus (9-10), high values of observed (0.762-0.798) and expected (0.770-0.791) heterozygosities, recent reduction of population size and gene flow. These findings constitute a baseline to measure potential changes in genetic diversity and structure of this commercially important species in a basin undergoing high anthropogenic activities.(AU)

El género de bagres neotropicales Pseudoplatystoma comprende ocho especies de gran tamaño, ampliamente distribuidas en las cuencas de Suramérica. La especie en peligro de extinción P. magdaleniatum es endémica de la Cuenca del Magdalena (Colombia), experimenta una alta presión pesquera y su genética poblacional es relativamente desconocida. Para estudiar el estado y estructura genética de P. magdaleniatum, se desarrollaron 25 loci microsatélites polimórficos especie específicos utilizando secuenciación de próxima generación y se evaluaron en muestras recolectadas en la Cuenca del Magdalena-Cauca. Con base en 15 loci, P. magdaleniatum mostró un alto número de alelos por locus (9-10), valores altos de heterocigosidad observada (0.762-0.798) y esperada (0.770-0.791), reducción reciente del tamaño poblacional y flujo génico. Estos hallazgos constituyen una línea de base para medir cambios potenciales en la diversidad y estructura genética de esta especie comercialmente importante en una cuenca sometida a altas actividades antropogénicas.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

Population genetics of the endangered catfish Pseudoplatystoma magdaleniatum (Siluriformes: Pimelodidae) based on species-specific microsatellite loci

The Neotropical catfish genus Pseudoplatystoma comprises eight species of large size, widely distributed in South American basins. The endangered species P. magdaleniatum is endemic to Magdalena basin (Colombia), experiences high fishing pressure and its population genetics is relatively unknown. To study the genetic status and structure of P. magdaleniatum, 25 species-specific polymorphic microsatellite loci were developed using next-generation sequencing and then tested in samples collected in the Magdalena-Cauca basin. Based on 15 of these loci, P. magdaleniatum showed a high number of alleles per locus (9-10), high values of observed (0.762-0.798) and expected (0.770-0.791) heterozygosities, recent reduction of population size and gene flow. These findings constitute a baseline to measure potential changes in genetic diversity and structure of this commercially important species in a basin undergoing high anthropogenic activities.(AU)

El género de bagres neotropicales Pseudoplatystoma comprende ocho especies de gran tamaño, ampliamente distribuidas en las cuencas de Suramérica. La especie en peligro de extinción P. magdaleniatum es endémica de la Cuenca del Magdalena (Colombia), experimenta una alta presión pesquera y su genética poblacional es relativamente desconocida. Para estudiar el estado y estructura genética de P. magdaleniatum, se desarrollaron 25 loci microsatélites polimórficos especie específicos utilizando secuenciación de próxima generación y se evaluaron en muestras recolectadas en la Cuenca del Magdalena-Cauca. Con base en 15 loci, P. magdaleniatum mostró un alto número de alelos por locus (9-10), valores altos de heterocigosidad observada (0.762-0.798) y esperada (0.770-0.791), reducción reciente del tamaño poblacional y flujo génico. Estos hallazgos constituyen una línea de base para medir cambios potenciales en la diversidad y estructura genética de esta especie comercialmente importante en una cuenca sometida a altas actividades antropogénicas.(AU)

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland

Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.(AU)

Microsatellite loci development for three catfish species from northwestern South America

The Neotropical catfish species Ageneiosus pardalis, Pimelodus grosskopfii, and Sorubim cuspicaudus are important fishery resources in Colombia that show historical declines in their capture. This study used next-generation sequencing with 454 FLX technology (Roche Applied Science) and bioinformatics analysis to develop between 18 and 24 microsatellite loci for these species. The novel microsatellite loci showed high values of polymorphic information content -PIC (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876), and the average number of alleles/locus ranged from 7-15 for A. pardalis, 9-30 for P. grosskopfii and 5-14 for S. cuspicaudus. The average observed and expected heterozygosities were respectively, 0.757 ± 0.035 and 0.834 ± 0.015 for A. pardalis; 0.596 ± 0.040 and 0.881 ± 0.009 for P. grosskopfii; and 0.747 ± 0.031 and 0.757 ± 0.025 for S. cuspicaudus. For future studies, these loci can be useful to estimate the genetic diversity and population structure in these three Neotropical catfishes.(AU)

Las especies de bagres neotropicales Ageneiosus pardalis, Pimelodus grosskopfii y Sorubim cuspicaudus, son importantes recursos pesqueros en Colombia y han mostrado disminuciones históricas en sus capturas. En este estudio se empleó la secuenciación genómica de próxima generación y análisis bioinformático para desarrollar entre 18 y 24 loci microsatélites para estas especies. Los loci microsatélites mostraron altos valores del contenido de información polimórfica CIP (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876) y el número promedio de alelos/locus mostró un rango de 7-15 para A. pardalis, 9-30 para P. grosskopfii y 5-14 para S. cuspicaudus. Los valores promedio de heterocigosidad observada y esperada fueron respectivamente 0.757 ± 0.035 y 0.834 ± 0.015 para A. pardalis; 0.596 ± 0.040 y 0.881 ± 0.009 para P. grosskopfii; y 0.747 ± 0.031y 0.757 ± 0.025 para S. cuspicaudus. Los loci microsatélites desarrollados en este trabajo pueden ser útiles para estimar la diversidad genética y la estructura poblacional de estos tres bagres neotropicales en estudios futuros.(AU)

Microsatellite loci development for three catfish species from northwestern South America

The Neotropical catfish species Ageneiosus pardalis, Pimelodus grosskopfii, and Sorubim cuspicaudus are important fishery resources in Colombia that show historical declines in their capture. This study used next-generation sequencing with 454 FLX technology (Roche Applied Science) and bioinformatics analysis to develop between 18 and 24 microsatellite loci for these species. The novel microsatellite loci showed high values of polymorphic information content -PIC (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876), and the average number of alleles/locus ranged from 7-15 for A. pardalis, 9-30 for P. grosskopfii and 5-14 for S. cuspicaudus. The average observed and expected heterozygosities were respectively, 0.757 ± 0.035 and 0.834 ± 0.015 for A. pardalis; 0.596 ± 0.040 and 0.881 ± 0.009 for P. grosskopfii; and 0.747 ± 0.031 and 0.757 ± 0.025 for S. cuspicaudus. For future studies, these loci can be useful to estimate the genetic diversity and population structure in these three Neotropical catfishes.(AU)

Las especies de bagres neotropicales Ageneiosus pardalis, Pimelodus grosskopfii y Sorubim cuspicaudus, son importantes recursos pesqueros en Colombia y han mostrado disminuciones históricas en sus capturas. En este estudio se empleó la secuenciación genómica de próxima generación y análisis bioinformático para desarrollar entre 18 y 24 loci microsatélites para estas especies. Los loci microsatélites mostraron altos valores del contenido de información polimórfica CIP (A. pardalis: 0.601-0.903, P. grosskopfii: 0.748-0.946 and S. cuspicaudus: 0.383-0.876) y el número promedio de alelos/locus mostró un rango de 7-15 para A. pardalis, 9-30 para P. grosskopfii y 5-14 para S. cuspicaudus. Los valores promedio de heterocigosidad observada y esperada fueron respectivamente 0.757 ± 0.035 y 0.834 ± 0.015 para A. pardalis; 0.596 ± 0.040 y 0.881 ± 0.009 para P. grosskopfii; y 0.747 ± 0.031y 0.757 ± 0.025 para S. cuspicaudus. Los loci microsatélites desarrollados en este trabajo pueden ser útiles para estimar la diversidad genética y la estructura poblacional de estos tres bagres neotropicales en estudios futuros.(AU)

Genomic characterization of Canine circovirus detected in a dog with intermittent hemorrhagic gastroenteritis in Brazil / Caracterização genômica do Canine circovirus detectado em um cão com gastroenterite hemorrágica intermitente no Brasil

Because Canine circovirus (CanineCV) is a new species of the genus Circovirus, several issues related to its epidemiology, pathogenesis and clinical disease remain unknown. Thus, this study aimed to perform the characterization of the first complete genome sequence of CanineCV detected in a dog with diarrhea in Brazil. A stool sample was collected of a ten-month-old female German Shepherd dog which had signs of intermittent hemorrhagic gastroenteritis, vomiting, and a history of eating raw pork. The complete CanineCV genome was sequenced by Next-Generation Sequencing. The sequence had 2,063 nucleotides, showed a typical genomic organization for circovirus, and was grouped with strain 214 described in the United States by phylogenetic analysis. One amino acid change was found in the replicase protein, and because of that it was considered unique to CanineCV. Therefore, the characterization of the complete genome of Brazilian CanineCV can be used in future studies of molecular epidemiology, pathogenesis and development of diagnostic tools for the prevention and control of this disease.(AU)

Como o Canine circovirus (CanineCV) é uma nova espécie do Gênero Circovirus, várias questões relacionadas com a sua epidemiologia, patogenia e doença clínica permanecem desconhecidas. Assim, este estudo objetivou realizar a caracterização da primeira sequência do genoma completo do CanineCV detectado em um cão com diarreia, no Brasil. Uma amostra de fezes foi coletada de um cão da raça Pastor Alemão, fêmea, 10 meses de idade, o qual tinha sinais de gastroenterite hemorrágica intermitente, vômito e uma história de ingestão de carne crua de porco. O genoma completo do CanineCV foi sequenciado pelo Sequenciamento de Nova Geração. A sequência tinha 2.063 nucleotídeos, apresentou uma organização genômica típica para um circovírus e foi agrupado com a cepa 214, descrita nos Estados Unidos pela análise filogenética. Uma mudança de aminoácido foi encontrada na proteína de replicação e por causa disso ela foi considerada única para o CanineCV. Portanto, a caracterização do genoma completo do CanineCV brasileiro pode ser utilizada em futuros estudos de epidemiologia molecular, patogenia e no desenvolvimento de ferramentas de diagnóstico para prevenção e controle desta doença.(AU)

Spatiotemporal expression pattern of alpha, beta, and gamma genes during BoHV-5 infection / Expressão temporal de genes alfa, beta e gama durante ainfecção pelo BoHV-5

Bovine herpesvirus 5 is an alphaherpesvirus that causes nonsuppurative meningoencephalitis in cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Although previous studies elucidated crucial aspects involved in the pathogenesis of the disease, there is a paucity of information regarding the molecular events contributing to infection and replication of BoHV-5. The objective of the present study was to determine the in vitro gene expression pattern of BoHV-5 (e.g., alpha, beta, and gamma genes) and host cells genes (GAPDH and 18S) over time utilizing different quantities of inoculated virus. Three BoHV-5 genes (bICP0, UL9, US4) and one structural bovine cell gene had their expression accessed by real-time PCR. While the expression of BoHV-5 genes increased during the course of infection, GAPDH gene expression decreased in the host cells, evidencing the effect of viral infection on the expression of bovine cell genes. The 18S ribosomal RNA (rRNA) gene was constitutively expressed throughout BoHV-5 infection. Our data clearly demonstrates that GAPDH gene should not be used as a reference gene in studies of BoHV-5 infection because it was influenced by viral infection. However, 18S rRNA was constitutively expressed and, therefore, is recommended for normalization of BoHV-5 infection studies in bovine cells. The expression of viral genes transcripts was not altered by increasing number of viral particles added to the culture. All viral genes included here demonstrated the same expression pattern over time and there was no difference in the expression of viral genes among the various time points. Our data show important differences comparing to classical studies regarding herpesvirus alpha, beta, and gamma genes expression. More research is necessary to improve our understanding about the BoHV-5 biology during infection. Studies employing next-generation sequencing (i.e., RNA-seq), using both in vitro and in vivo models, would be the next logical step to grasp the virus and host cell's transcriptome changes over the course of infection.(AU)

Herpesvirus bovino 5 é um alfaherpesvírus causador de meningoencefalite não supurativa em bovinos. Esta doença possui ocorrência natural em surtos ou casos isolados, associadas a baixa morbidade e alta letalidade. Embora estudos anteriores tenham elucidado aspectos relacionados a patogenia da doença, há uma lacuna de conhecimento relacionado aos eventos moleculares que contribuem para a infecção e replicação do BoHV-5. O objetivo do presente estudo foi determinar a expressão gênica in vitro de genes virais (i.e., alfa, beta e gama) e das células hospedeiras (GAPDH e 18S) durante a infecção considerando diferentes momentos de infecção e quantidade de vírus utilizado. Três genes do BoHV-5 (bICP0, UL9, US4), um gene estrutural (GAPDH) e um gene constitutivo (18S) da célula bovina tiveram suas expressões avaliadas por PCR quantitativa (qPCR). Enquanto os genes virais tiveram sua expressão aumentada ao longo do tempo de infecção, o gene hospedeiro teve sua expressão diminuída, demonstrando a ação do vírus na expressão gênica de células bovinas in vitro. O gene constitutivo 18S teve sua expressão mantida durante todos os momentos do experimento. Nossos resultados claramente demonstraram que o GAPDH não deve ser usado como gene de referência em estudos com infecção por BoHV-5 pois é influenciado pela infecção viral. Entretanto, o 18S rRNA foi constitutivamente expresso e pode ser recomendado para normalização em células bovinas infectadas pelo vírus. A expressão de mRNA viral não foi alterada pela quantidade de vírus usada. Todos os genes virais demonstraram o mesmo padrão de expressão ao longo do tempo de infecção. Nossos resultados trazem importantes diferenças comparando aos estudos clássicos que avaliaram a expressão de genes alfa, beta e gama. Mais estudos são necessários para aumentar o conhecimento da biologia molecular do BoHV-5. Estudo utilizando sequenciamento de última geração (i.e., RNA-seq), usando modelos in vitro e in vivo, aparentam ser o próximo passo lógico para acessar as alterações do transcriptoma do hospedeiro e viral ao longo do curso da infecção.(AU)

Spatiotemporal expression pattern of alpha, beta, and gamma genes during BoHV-5 infection / Expressão temporal de genes alfa, beta e gama durante ainfecção pelo BoHV-5

Bovine herpesvirus 5 is an alphaherpesvirus that causes nonsuppurative meningoencephalitis in cattle. This disease occurs naturally in either outbreaks or isolated cases, and exhibits low morbidity and high lethality. Although previous studies elucidated crucial aspects involved in the pathogenesis of the disease, there is a paucity of information regarding the molecular events contributing to infection and replication of BoHV-5. The objective of the present study was to determine the in vitro gene expression pattern of BoHV-5 (e.g., alpha, beta, and gamma genes) and host cells genes (GAPDH and 18S) over time utilizing different quantities of inoculated virus. Three BoHV-5 genes (bICP0, UL9, US4) and one structural bovine cell gene had their expression accessed by real-time PCR. While the expression of BoHV-5 genes increased during the course of infection, GAPDH gene expression decreased in the host cells, evidencing the effect of viral infection on the expression of bovine cell genes. The 18S ribosomal RNA (rRNA) gene was constitutively expressed throughout BoHV-5 infection. Our data clearly demonstrates that GAPDH gene should not be used as a reference gene in studies of BoHV-5 infection because it was influenced by viral infection. However, 18S rRNA was constitutively expressed and, therefore, is recommended for normalization of BoHV-5 infection studies in bovine cells. The expression of viral genes transcripts was not altered by increasing number of viral particles added to the culture. All viral genes included here demonstrated the same expression pattern over time and there was no difference in the expression of viral genes among the various time points. Our data show important differences comparing to classical studies regarding herpesvirus alpha, beta, and gamma genes expression. More research is necessary to improve our understanding about the BoHV-5 biology during infection. Studies employing next-generation sequencing (i.e., RNA-seq), using both in vitro and in vivo models, would be the next logical step to grasp the virus and host cell's transcriptome changes over the course of infection.(AU)

Herpesvirus bovino 5 é um alfaherpesvírus causador de meningoencefalite não supurativa em bovinos. Esta doença possui ocorrência natural em surtos ou casos isolados, associadas a baixa morbidade e alta letalidade. Embora estudos anteriores tenham elucidado aspectos relacionados a patogenia da doença, há uma lacuna de conhecimento relacionado aos eventos moleculares que contribuem para a infecção e replicação do BoHV-5. O objetivo do presente estudo foi determinar a expressão gênica in vitro de genes virais (i.e., alfa, beta e gama) e das células hospedeiras (GAPDH e 18S) durante a infecção considerando diferentes momentos de infecção e quantidade de vírus utilizado. Três genes do BoHV-5 (bICP0, UL9, US4), um gene estrutural (GAPDH) e um gene constitutivo (18S) da célula bovina tiveram suas expressões avaliadas por PCR quantitativa (qPCR). Enquanto os genes virais tiveram sua expressão aumentada ao longo do tempo de infecção, o gene hospedeiro teve sua expressão diminuída, demonstrando a ação do vírus na expressão gênica de células bovinas in vitro. O gene constitutivo 18S teve sua expressão mantida durante todos os momentos do experimento. Nossos resultados claramente demonstraram que o GAPDH não deve ser usado como gene de referência em estudos com infecção por BoHV-5 pois é influenciado pela infecção viral. Entretanto, o 18S rRNA foi constitutivamente expresso e pode ser recomendado para normalização em células bovinas infectadas pelo vírus. A expressão de mRNA viral não foi alterada pela quantidade de vírus usada. Todos os genes virais demonstraram o mesmo padrão de expressão ao longo do tempo de infecção. Nossos resultados trazem importantes diferenças comparando aos estudos clássicos que avaliaram a expressão de genes alfa, beta e gama. Mais estudos são necessários para aumentar o conhecimento da biologia molecular do BoHV-5. Estudo utilizando sequenciamento de última geração (i.e., RNA-seq), usando modelos in vitro e in vivo, aparentam ser o próximo passo lógico para acessar as alterações do transcriptoma do hospedeiro e viral ao longo do curso da infecção.(AU)

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.