Antimicrobial Resistance Profile and ExPEC Virulence Potential in Commensal Escherichia coli of Multiple Sources.

We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants.

Isolation, culture, and characterization of chicken intestinal epithelial cells.

BACKGROUND: Enterocytes exert an absorptive and protective function in the intestine, and they encounter many different challenging factors such as feed, bacteria, and parasites. An intestinal epithelial in vitro model can help to understand how enterocytes are affected by these factors and contribute to the development of strategies against pathogens. RESULTS: The present study describes a novel method to culture and maintain primary chicken enterocytes and their characterization by immunofluorescence and biomolecular approaches. Starting from 19-day-old chicken embryos it was possible to isolate viable intestinal cell aggregates that can expand and produce a self-maintaining intestinal epithelial cell population that survives until 12 days in culture. These cells resulted positive in immunofluorescence to Cytokeratin 18, Zonula occludens 1, Villin, and Occludin that are common intestinal epithelial markers, and negative to Vimentin that is expressed by endothelial cells. Cells were cultured also on Transwell® permeable supports and trans-epithelial electrical resistance, was measured. This value gradually increased reaching 64 Ω*cm2 7 days after seeding and it remained stable until day 12. CONCLUSIONS: Based on these results it was confirmed that it is possible to isolate and maintain chicken intestinal epithelial cells in culture and that they can be suitable as in vitro intestinal model for further studies.

In vitro anticoccidial activity of thymol, carvacrol, and saponins.

The anticoccidial activity of thymol, carvacrol, and saponins was assessed in an in vitro model of coccidiosis. Eimeria spp. sporozoites were collected from field samples, characterized, and used for 2 different invasion assays on Madin-Darby Bovine Kidney cells (MDBK). The cells were challenged with 5 × 104 sporozoites without (control) or with various treatments: saponins (10 ppm), thymol, and carvacrol (7 ppm each) or a combination of saponins, thymol, and carvacrol at 2 doses; MIX 1 (saponins 5 ppm, thymol 3.5 ppm, and carvacrol 3.5 ppm) and MIX 2 (saponins 10 ppm, thymol 7 ppm, and carvacrol 7 ppm). The treated cells were incubated at 37°C for 24 h (invasion assay 1) and for 2, 24, and 48 h (invasion assay 2). The efficiency of invasion was determined by counting the sporozoites left in the supernatant that were not able to invade the cells, whereas intracellular Eimeria DNA was detected by qPCR to confirm the data. Data were analyzed with ANOVA, and differences were considered significant when P value was ≤0.05. Data from invasion assay 1 showed that the thymol and carvacrol-containing blends significantly reduced invasion, especially in combination with saponins at the highest dose. Saponins alone did not have a strong inhibiting activity but acted synergistically with the other molecules. Interestingly, in invasion assay 2, it was found that the effect of the highest dose of the blend of saponins, thymol, and carvacrol was already visible at 2 h postinfection, whereas the other treatments were significantly successful at 24 h postinfection. The invasion assay protocol was designed to screen molecules in vitro starting from field fecal samples, and it can represent a potential tool in Eimeria research. Moreover, this study shows that invasion in MDBK cells by Eimeria sporozoites is inhibited in presence of thymol, carvacrol, and saponins, thus highlighting the anticoccidial potential of these compounds.

Phylodynamic analysis and evaluation of the balance between anthropic and environmental factors affecting IBV spreading among Italian poultry farms.

Infectious bronchitis virus (IBV) control is mainly based on wide vaccine administration. Although effective, its efficacy is not absolute, the viral circulation is not prevented and some side effects cannot be denied. Despite this, the determinants of IBV epidemiology and the factors affecting its circulation are still largely unknown and poorly investigated. In the present study, 361 IBV QX (the most relevant field genotype in Italy) sequences were obtained between 2012 and 2016 from the two main Italian integrated poultry companies. Several biostatistical and bioinformatics approaches were used to reconstruct the history of the QX genotype in Italy and to assess the effect of different environmental, climatic and social factors on its spreading patterns. Moreover, two structured coalescent models were considered in order to investigate if an actual compartmentalization occurs between the two integrated poultry companies and the role of a third "ghost" deme, representative of minor industrial poultry companies and the rural sector. The obtained results suggest that the integration of the poultry companies is an effective barrier against IBV spreading, since the strains sampled from the two companies formed two essentially-independent clades. Remarkably, the only exceptions were represented by farms located in the high densely populated poultry area of Northern Italy. The inclusion of a third deme in the model revealed the likely role of other poultry companies and rural farms (particularly concentrated in Northern Italy) as sources of strain introduction into one of the major poultry companies, whose farms are mainly located in the high densely populated poultry area of Northern Italy. Accordingly, when the effect of different environmental and urban parameters on IBV geographic spreading was investigated, no factor seems to contribute to IBV dispersal velocity, being poultry population density the only exception. Finally, the different viral population pattern observed in the two companies over the same time period supports the pivotal role of management and control strategies on IBV epidemiology. Overall, the present study results stress the crucial relevance of human action rather than environmental factors, highlighting the direct benefits that could derive from improved management and organization of the poultry sector on a larger scale.

Cauliflower Mosaic Virus TAV, a Plant Virus Protein That Functions like Ribonuclease H1 and is Cytotoxic to Glioma Cells.

Recent comparisons between plant and animal viruses reveal many common principles that underlie how all viruses express their genetic material, amplify their genomes, and link virion assembly with replication. Cauliflower mosaic virus (CaMV) is not infectious for human beings. Here, we show that CaMV transactivator/viroplasmin protein (TAV) shares sequence similarity with and behaves like the human ribonuclease H1 (RNase H1) in reducing DNA/RNA hybrids detected with S9.6 antibody in HEK293T cells. We showed that TAV is clearly expressed in the cytosol and in the nuclei of transiently transfected human cells, similar to its distribution in plants. TAV also showed remarkable cytotoxic effects in U251 human glioma cells in vitro. These characteristics pave the way for future analysis on the use of the plant virus protein TAV, as an alternative to human RNAse H1 during gene therapy in human cells.

Marek's disease viruses circulating in commercial poultry in Italy in the years 2015-2018 are closely related by their meq gene phylogeny.

Marek's disease (MD) is a lymphoproliferative disease important to the poultry industry worldwide; it is caused by Gallid alphaherpesvirus 2 (GaHV-2). The virulence of GaHV-2 isolates has shifted over the years from mild to virulent, very virulent and very virulent +. Nowadays the disease is controlled by vaccination, but field strains of increased virulence are emerging worldwide. Economic losses due to MD are mostly associated with its acute form, characterized by visceral lymphomas. The present study aimed to molecularly classify a group of 13 GaHV-2 strains detected in vaccinated Italian commercial chicken flocks during acute MD outbreaks, and to scrutinize the ability of predicting GaHV-2 virulence, according to the meq gene sequence. The full-length meq genes were amplified, and the obtained amino acid (aa) sequences were analysed, focusing mainly on the number of stretches of four proline molecules (PPPP) within the transactivation domain. Phylogenetic analysis was carried out with the Maximum Likelihood method using the obtained aa sequences, and the sequences of Italian strains detected in backyard flocks and of selected strains retrieved from GenBank. All the analysed strains showed 100% sequence identity in the meq gene, which encodes a Meq protein of 339 aa. The Meq protein includes four PPPP motifs in the transactivation domain and an interruption of a PPPP motif due to a proline-to-serine substitution at position 218. These features are typically encountered in highly virulent isolates. Phylogenetic analysis revealed that the analysed strains belonged to a cluster that includes high-virulence GaHV-2 strains detected in Italian backyard flocks and a hypervirulent Polish strain. Our results support the hypothesis that the virulence of field isolates can be suggested by meq aa sequence analysis.

Molecular characterization of a Marek's disease virus strain detected in tumour-bearing turkeys.

Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2), which primarily affects chickens. However, the virus is also able to induce tumours in turkeys, albeit less frequently than in chickens. This study reports the molecular characterization of a GaHV-2 strain detected in a flock of Italian meat-type turkeys exhibiting visceral lymphomas. Sequencing and phylogenetic analysis of the meq gene revealed that the turkey GaHV-2 has molecular features of high virulence and genetic similarity with GaHV-2 strains recently detected in Italian commercial and backyard chickens. GaHV-2 is ubiquitous among chickens despite vaccination, and chicken-to-turkey transmission is hypothesized due to the presence of broilers in neighbouring pens.RESEARCH HIGHLIGHTS A GaHV-2 strain from Italian turkeys was molecularly characterized.The turkey strain presented molecular characteristics of high virulence in its meq gene.The turkey strain was closely related to previously detected chicken strains.

Effect of Chestnut Tannins and Short Chain Fatty Acids as Anti-Microbials and as Feeding Supplements in Broilers Rearing and Meat Quality.

Chestnut tannins (CT) and saturated short medium chain fatty acids (SMCFA) are valid alternatives to contrast the growth of pathogens in poultry rearing, representing a valid alternative to antibiotics. However, the effect of their blends has never been tested. Two blends of CT extract and Sn1-monoglycerides of SMCFA (SN1) were tested in vitro against the proliferation of Clostridium perfringens, Salmonella typhymurium, Escherichia coli, Campylobacter jejuni. The tested concentrations were: 3.0 g/kg of CT; 3.0 g/kg of SN1; 2.0 g/kg of CT and 1.0 g/kg of SN1; 1.0 g/kg of CT and 2.0 g/kg of SN1. Furthermore, their effect on broiler performances and meat quality was evaluated in vivo: one-hundred Ross 308 male birds were fed a basal diet with no supplement (control group) or supplemented with CT or SN1 or their blends at the same concentration used in the in vitro trial. The in vitro assay confirmed the effectiveness of the CT and SN1 mixtures in reducing the growth of the tested bacteria while the in vivo trial showed that broiler performances, animal welfare and meat quality were not negatively affected by the blends, which could be a promising alternative in replacing antibiotics in poultry production.

In Vitro Antimicrobial Activity of Essential Oils Against Salmonella enterica Serotypes Enteritidis and Typhimurium Strains Isolated from Poultry.

Salmonella enterica serotype Enteritidis and S. enterica serotype Typhimurium are frequently present among poultry and are associated with outbreaks of human salmonellosis. The study investigated the in vitro antimicrobial activity of essential oils (EOs) obtained from Aloysia triphylla, Cinnamomum zeylanicum, Cymbopogon citratus, Litsea cubeba, Mentha piperita, Syzygium aromaticum against S. Enteritidis and S. Thyphimurium strains previously isolated from poultry. A 1:1 mixture of C. zeylanicum and S. aromaticum was also tested. The activity of all compounds was evaluated against the yeast Saccharomyces cerevisiae, commonly used as probiotic. The highest antibacterial activity was observed for C. zeylanicum (minimum inhibitory concentrations (MICs) ranging from 1.26 mg/mL to 0.63 mg/mL), S. aromaticum (MICs from 2.637 mg/mL to 0.164 mg/mL) and the mixture (MICs from 1.289 mg/mL to 0.322 mg/mL). No activity was recorded against S. cerevisiae. The results suggest a possible use of C. zeylanicum and S. aromaticum, alone or in combination, in the farm environment for disinfection and in poultry diet, combined with S. cerevisiae administration, for an integrated approach to avoid Salmonella intestinal colonization.

GI-16 lineage (624/I or Q1), there and back again: The history of one of the major threats for poultry farming of our era.

The genetic variability of Infectious bronchitis virus (IBV) is one of the main challenges for its control, hindering not only the development of effective vaccination strategies but also its classification and, consequently, epidemiology understanding. The 624/I and Q1 genotypes, now recognized to be part of the GI-16 lineage, represent an excellent example of the practical consequences of IBV molecular epidemiology limited knowledge. In fact, being their common origin unrecognized for a long time, independent epidemiological pictures were drawn for the two genotypes. To fix this misinterpretation, the present study reconstructs the history, population dynamics and spreading patterns of GI-16 lineage as a whole using a phylodynamic approach. A collection of worldwide available hypervariable region 1 and 2 (HVR12) and 3 (HVR3) sequences of the S1 protein was analysed together with 258 HVR3 sequences obtained from samples collected in Italy (the country where this genotype was initially identified) since 1963. The results demonstrate that after its emergence at the beginning of the XX century, GI-16 was able to persist until present days in Italy. Approximately in the late 1980s, it migrated to Asia, which became the main nucleus for further spreading to Middle East, Europe and especially South America, likely through multiple introduction events. A remarkable among-country diffusion was also demonstrated in Asia and South America. Interestingly, although most of the recent Italian GI-16 strains originated from ancestral viruses detected in the same country, a couple were closely related to Chinese ones, supporting a backward viral flow from China to Italy. Besides to the specific case-study results, this work highlights the misconceptions that originate from the lack of a unified nomenclature and poor molecular epidemiology data generation and sharing. This shortcoming appears particularly relevant since the described scenario could likely be shared by many other IBV genotypes and pathogens in general.

Some pathogenic characters of paratyphoid Salmonella enterica strains isolated from poultry.

OBJECTIVE: To investigate some pathogenic characters of Salmonella enterica strains isolated from poultry. METHODS: Twenty-three genetically distinct Salmonella enterica strains, of different serovars and pulsotype, were examined for virulence traits. Resistance to gastric acid environment was estimated by measuring the percentage of survived bacterial cells after exposure for 2 h to a synthetic gastric juice. Strains were analyzed with PCR for the presence of the following virulence genes: mgtC and rhuM located on SPI-3, sopB and pipB located on SPI-5, Salmonella virulence plasmid (spv) R (spvR), spvB and spvC located on Salmonella plasmid virulence and sodCI, sopE, and gipA located on prophage. Finally, resistance to 21 antibiotics was tested with Kirby-Bauer method. RESULTS: A percentage of 82.60% of strains were resistant to gastric environment after induction and 60.87% of the strains exhibited constitutive resistance too. Nineteen different virulence profiles were detected. The phage related genes sodCI and sopE and the plasmid mediated operon spvR, spvB and spvC (spvRBC) were detected in 82.60%, 47.82% and 52.17% of strains, respectively. Typhimurium and Enteritidis strains showed the highest number of virulence genes. Twenty-one different antibiotic resistance profiles were obtained and two isolates (Typhimurium and Enteritidis) resulted sensible to all the tested molecules. The ampicillin, streptomycin, sulfonamide and tetracycline resistance profile was detected in seven isolates (30.43%). CONCLUSION: Our results show that paratyphoid Salmonella strains with several characters of pathogenicity, that may be cause of severe pathology in animals and humans, are circulating among poultry.

Think globally, act locally: Phylodynamic reconstruction of infectious bronchitis virus (IBV) QX genotype (GI-19 lineage) reveals different population dynamics and spreading patterns when evaluated on different epidemiological scales.

Infectious bronchitis virus (IBV) represents one of the poultry industry major threats, particularly in high density producing countries. The emergence and spread of new IBV genotypes have frustrated the various disease control efforts implemented over time. Despite that, few comprehensive and large scale studies have been performed to understand the international and local spreading dynamics of this virus. In the present work, these phenomena were evaluated by implementing a Bayesian phylodynamic approach to reconstruct the epidemiological patterns and population history of the QX genotype (currently renamed GI-19 lineage), the most relevant IBV lineage of the Old-World. Our analysis, based on 807 partial S1 sequences of strains collected from 18 countries between 1993 and 2015, demonstrates that this genotype originated in China well before its first identification. After a prolonged local circulation, it started spreading to other European, Asian and Middle East countries in successive waves, which were mirrored by concomitant fluctuations in viral population size. Interestingly, the within-Europe spread was characterized by a higher estimated migration rate compared with the inter-continental one, potentially reflecting the closer geographic and economic relationships among these countries. Nevertheless, the colonization of new states by the GI-19 lineage appeared to occur mostly by single introduction events in both intra and inter-continental spread, likely because of epidemiological factor and health policy combination which seems to prevent the frequent introduction and mixing of different strains. On the other hand, the within Italy QX circulation reconstruction showed a much more intricate connection network among different locations, evidencing the difficulty in controlling IBV spread especially in highly densely poultry populated areas. The presence of several well supported epidemiological links among distantly related Italian regions testifies that animal transportation and indirect transmission routes rather than local airborne diffusion contribute to the QX success and persistence at local scale. Globally, the spreading dynamics and evolution of the QX genotype were reconstructed from its very origin to nowadays, demonstrating the need of more effective direct control measures, particularly within each country. Unfortunately, the incompleteness of available molecular epidemiology data represents an insurmountable limit which leaves many questions currently unsolved, thus highlighting the compulsoriness of a structured monitoring and data sharing system implementation.

Genetic Diversity of Highly Pathogenic Avian Influenza A(H5N8/H5N5) Viruses in Italy, 2016-17.

In winter 2016-17, highly pathogenic avian influenza A(H5N8) and A(H5N5) viruses of clade 2.3.4.4 were identified in wild and domestic birds in Italy. We report the occurrence of multiple introductions and describe the identification in Europe of 2 novel genotypes, generated through multiple reassortment events.

Unexpected Interfarm Transmission Dynamics during a Highly Pathogenic Avian Influenza Epidemic.

UNLABELLED: Next-generation sequencing technology is now being increasingly applied to study the within- and between-host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep-sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel (i) the epidemic virus population diversity, (ii) the evolution of virus pathogenicity, and (iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the cocirculation in the index case of two viral strains showing a different insertion at the hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess interfarm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed interfarm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain. IMPORTANCE: The within- and between-host evolutionary dynamics of a highly pathogenic avian influenza (HPAI) strain during a naturally occurring epidemic is currently poorly understood. Here, we perform for the first time an in-depth sequence analysis of all the samples collected during a HPAI epidemic and demonstrate the importance to complement outbreak investigations with genetic data to reconstruct the transmission dynamics of the viruses and to evaluate the within- and between-farm genetic diversity of the viral population. We show that the evolutionary transition from the low pathogenic form to the highly pathogenic form occurred within the first infected flock, where we identified haplotypes with hemagglutinin cleavage site of different lengths. We also identify the index case as the major source of virus, indicating that prompt application of depopulation measures is essential to limit virus spread to other farms.

Full Genome Sequence-Based Comparative Study of Wild-Type and Vaccine Strains of Infectious Laryngotracheitis Virus from Italy.

Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980, 2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and may be involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains.

Cannabidiol, a non-psychoactive cannabinoid compound, inhibits proliferation and invasion in U87-MG and T98G glioma cells through a multitarget effect.

In the present study, we found that CBD inhibited U87-MG and T98G cell proliferation and invasiveness in vitro and caused a decrease in the expression of a set of proteins specifically involved in growth, invasion and angiogenesis. In addition, CBD treatment caused a dose-related down-regulation of ERK and Akt prosurvival signaling pathways in U87-MG and T98G cells and decreased hypoxia inducible factor HIF-1α expression in U87-MG cells. Taken together, these results provide new insights into the antitumor action of CBD, showing that this cannabinoid affects multiple tumoral features and molecular pathways. As CBD is a non-psychoactive phytocannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anti-cancer drug in the management of gliomas.

Exploring the larval transcriptome of the common sole (Solea solea L.).

BACKGROUND: The common sole (Solea solea) is a promising candidate for European aquaculture; however, the limited knowledge of the physiological mechanisms underlying larval development in this species has hampered the establishment of successful flatfish aquaculture. Although the fact that genomic tools and resources are available for some flatfish species, common sole genomics remains a mostly unexplored field. Here, we report, for the first time, the sequencing and characterisation of the transcriptome of S. solea and its application for the study of molecular mechanisms underlying physiological and morphological changes during larval-to-juvenile transition. RESULTS: The S. solea transcriptome was generated from whole larvae and adult tissues using the Roche 454 platform. The assembly process produced a set of 22,223 Isotigs with an average size of 726 nt, 29 contigs and a total of 203,692 singletons. Of the assembled sequences, 75.2% were annotated with at least one known transcript/protein; these transcripts were then used to develop a custom oligo-DNA microarray. A total of 14,674 oligonucleotide probes (60 nt), representing 12,836 transcripts, were in situ synthesised onto the array using Agilent non-contact ink-jet technology. The microarray platform was used to investigate the gene expression profiles of sole larvae from hatching to the juvenile form. Genes involved in the ontogenesis of the visual system are up-regulated during the early stages of larval development, while muscle development and anaerobic energy pathways increase in expression over time. The gene expression profiles of key transcripts of the thyroid hormones (TH) cascade and the temporal regulation of the GH/IGF1 (growth hormone/insulin-like growth factor I) system suggest a pivotal role of these pathways in fish growth and initiation of metamorphosis. Pre-metamorphic larvae display a distinctive transcriptomic landscape compared to previous and later stages. Our findings highlighted the up-regulation of gene pathways involved in the development of the gastrointestinal system as well as biological processes related to folic acid and retinol metabolism. Additional evidence led to the formation of the hypothesis that molecular mechanisms of cell motility and ECM adhesion may play a role in tissue rearrangement during common sole metamorphosis. CONCLUSIONS: Next-generation sequencing provided a good representation of the sole transcriptome, and the combination of different approaches led to the annotation of a high number of transcripts. The construction of a microarray platform for the characterisation of the larval sole transcriptome permitted the definition of the main processes involved in organogenesis and larval growth.

Cannabidiol as potential anticancer drug.

Over the past years, several lines of evidence support an antitumourigenic effect of cannabinoids including Δ(9)-tetrahydrocannabinol (Δ(9)-THC), synthetic agonists, endocannabinoids and endocannabinoid transport or degradation inhibitors. Indeed, cannabinoids possess anti-proliferative and pro-apoptotic effects and they are known to interfere with tumour neovascularization, cancer cell migration, adhesion, invasion and metastasization. However, the clinical use of Δ(9)-THC and additional cannabinoid agonists is often limited by their unwanted psychoactive side effects, and for this reason interest in non-psychoactive cannabinoid compounds with structural affinity for Δ(9)-THC, such as cannabidiol (CBD), has substantially increased in recent years. The present review will focus on the efficacy of CBD in the modulation of different steps of tumourigenesis in several types of cancer and highlights the importance of exploring CBD/CBD analogues as alternative therapeutic agents.

Multilocus variable-number of tandem-repeats analysis of Salmonella enterica serotype Gallinarum and comparison with pulsed-field gel electrophoresis genotyping.

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, a severe disease of poultry, responsible for heavy economic losses. Epidemiologic investigation of fowl typhoid significantly benefits from molecular typing tools, RAPD and PFGE have been proposed for this purpose. PFGE, a well established technique, is still the gold standard among typing methods for most bacteria, including salmonella. Nevertheless, it has some limitations regarding execution and reproducibility, in particular it is labour intensive and requires good technical expertise. Furthermore, it needs accurate standardization and results can be ambiguous to interpret. Such limitations can hamper reproducibility and transfer of results. As a possible alternative to PFGE, multilocus variable-number of tandem-repeats analysis (MLVA) has recently emerged as an effective genotyping method for many bacterial pathogens showing high discriminatory power associated to robustness. We developed a six-loci MLVA protocol for Salmonella Gallinarum and compared it to PFGE performed with SpeI, XbaI and NotI on fifty isolates. The proposed MLVA has a high discriminatory power, equivalent to that of the three-enzyme PFGE (Simpson's index 0.94 for MLVA, 0.93 for three-enzyme PFGE) but it is simpler to perform and straightforward in genotype identification, allowing unambiguous exchange of results. Stability of selected VNTR loci, assessed in vitro and in vivo, is good but not absolute, reflecting the sensitivity of MLVA to detect evolutionary changes of bacteria. Clustering of the isolates as determined by MLVA typing is substantially confirmed by PFGE.

Molecular mechanisms involved in the antitumor activity of cannabinoids on gliomas: role for oxidative stress.

Cannabinoids, the active components of Cannabis sativa, have been shown to exert antiproliferative and proapoptotic effects on a wide spectrum of tumor cells and tissues. Of interest, cannabinoids have displayed great potency in reducing the growth of glioma tumors, one of the most aggressive CNS tumors, either in vitro or in animal experimental models curbing the growth of xenografts generated by subcutaneous or intrathecal injection of glioma cells in immune-deficient mice. Cannabinoids appear to be selective antitumoral agents as they kill glioma cells without affecting the viability of non-transformed cells. This review will summarize the anti-cancer properties that cannabinoids exert on gliomas and discuss their potential action mechanisms that appear complex, involving modulation of multiple key cell signaling pathways and induction of oxidative stress in glioma cells.