SURVEY ON V. CHOLERAE, V. VULNIFICUS AND V. PARAHAEMOLYTICUS IN BIVALVE MOLLUSCS OF THE ADRIATIC SEA AND PROPOSAL OF AN ANALYTICAL PROTOCOL

Bivalve molluscs from Adriatic sea were analyzed for V. parahaemolyticus, V. cholerae e V. vulnificus presence. The isolates on TCBS Agar and m-CPC Agar were selected on the basis of a new biochemical screening, that showed a good performance, because among 2344 strains from primary culture only 237 (10%) were presumptively assigned to the species of interest. The PCR analyses was performed for the target genes toxR hlyA, ctxA, tcpI (V. cholerae), toxR, tl, tdh, trh (V. parahaemolyticus), vvhA and viuB (V. vulnificus). Among the 9 strains confirmed to belong to V. parahaemolyticus specie, 6 were sucrose positive. On 215 samples of molluscs only 5 resulted positive for V. parahaemolyticus being toxR+, tl+, although non pathogenic (tdh-, trh-), and none for V. cholerae e V. vulnificus

Group polarization effect on decisions by selected Kenyan secondary school disciplinary panels

This study investigated social group phenomenon of group polarization effects on disciplinary hearing decisions in selected Kenyan secondary school. The participants were 78 school personnel (females = 42%and males 58%) from ten secondary schools with both unisex (n = 39) and co-educational schools (n = 39). Both quantitative and qualitative data were collected and analyzed. The results suggested group polarization effects in disciplinary hearing decisions, in that there were shifts from pre to post-disciplinary hearing decisions. Persuasive arguments and social comparisons significantly influenced group polarization decisions.Department of HE and Training approved lis

Characterization and Whole Genome Analysis of Human Papillomavirus Type 16 E1-1374^63nt Variants

Background. The variation of the most common Human papillomavirus (HPV) type found in cervical cancer, the HPV16, has been extensively investigated in almost all viral genes. The E1 gene variation, however, has been rarely studied. The main objective of the present investigation was to analyze the variability of the E6 and E1 genes, focusing on the recently identified E1-1374^63nt variant. Methodology/Principal Findings. Variation within the E6 of 786 HPV16 positive cervical samples was analyzed using high-resolution melting, while the E1-1374^63nt duplication was assayed by PCR. Both techniques were supplemented with sequencing. The E1-1374^63nt duplication was linked with the E-G350 and the E-C109/G350 variants. In comparison to the referent HPV16, the E1-1374^63nt E-G350 variant was significantly associated with lower grade cervical lesions (p=0.029), while the E1-1374^63nt E-C109/G350 variant was equally distributed between high and low grade lesions. The E1-1374^63nt variants were phylogenetically closest to E-G350 variant lineage (A2 sub-lineage based on full genome classification). The major differences between E1-1374^63nt variants were within the LCR and the E6 region. On the other hand, changes within the E1 region were the major differences from the A2 sub-lineage, which has been historically but inconclusively associated with high grade cervical disease. Thus, the shared variations cannot explain the particular association of the E1-1374^63nt variant with lower grade cervical lesions. Conclusions/Significance. The E1 region has been thus far considered to be well conserved among all HPVs and therefore uninteresting for variability studies. However, this study shows that the variations within the E1 region could possibly affect cervical disease, since the E1-1374^63nt E-G350 variant is significantly associated with lower grade cervical lesions, in comparison to the A1 and A2 sub-lineage variants. Furthermore, it appears that the silent variation 109T>C of the E-C109/G350 variant might have a significant role in the viral life cycle and warrants further study

Impacts of soil conditions and light availability on natural regeneration of Norway spruce Picea abies (L.) H. Karst. in low-elevation mountain forests

& Key message Natural regeneration of P. abies (L.) H. Karst. may reach high densities in lower mountain elevations. The highest densities were found in sites with moderate light availability, with low pH, and not near the riverbank. However, age-height classes differed in the predicted magnitude of response, but were consistent in response directions. Mosses and understory species typical of coniferous forests were positively correlated with regeneration density. & Context Norway spruce Picea abies (L.) H. Karst. in Central Europe is at risk under climate change scenarios, particularly in mountain regions. Little is known about the impact of environmental factors on the natural regeneration of P. abies in lowelevation mountain forests. & Aims We aimed to assess impacts of distance from the riverbank, soil pH, and light availability on natural P. abies regeneration. We hypothesized that (1) natural P. abiesregeneration would depend on light availability and soil pH and (2) there are understory plant species which may indicate the microsites suitable for natural regeneration of P. abies. & Methods The study was conducted in the Stołowe Mountains National Park (SW Poland, 600–800 m a.s.l.). We established 160 study plots (25 m2 ) for natural regeneration, light availability, soil pH, and understory vegetation assessment

Effetto virucida di un disinfettante commerciale contro Betanodavirus.

L\u2019acquacoltura \ue8 a livello mondiale un settore che sta registrando un costante e continuo aumento delle produzioni con conseguente richiesta di implementazione degli standard igienici e di efficaci prodotti e protocolli di disinfezione al fine di ridurre i rischi sanitari legati alla diffusione delle malattie infettive. Nella realt\ue0 europea e italiana il branzino (Dicentrarchus labrax) \ue8 una delle specie marine con pi\uf9 elevato interesse per l\u2019acquacoltura. Fra le malattie infettive che maggiormente affliggono questa produzione si registra l\u2019Encefalo Retinopatia Virale (ERV) da Betanodavirus. Questa patologia \ue8 responsabile di sintomatologia nervosa e mortalit\ue0 con conseguenti elevate perdite economiche. Attualmente non sono disponibili terapie e vaccini che permettano un adeguato controllo dell\u2019ERV, per cui la prevenzione della malattia \ue8 affidata al mantenimento di corrette procedure igienico-sanitarie e alla corretta gestione dell\u2019allevamento che si basa su un\u2019approfondita conoscenza delle caratteristiche del patogeno e sulla disponibilit\ue0 di prodotti e protocolli di disinfezione per il controllo dello stesso. Al fine di ampliare le conoscenze sull\u2019efficacia di prodotti disinfettanti verso Betanodavirus, sono state intraprese, in questo studio, delle prove in vitro per valutare l\u2019attivit\ue0 virucida specifica di un prodotto commerciale denominato Virkon\uaeS gi\ue0 ampiamente utilizzato in ambito zootecnico. L\u2019attivit\ue0 virucida \ue8 stata valutata tenendo conto di diversi tempi di contatto, diverse temperature di incubazione ed in presenza di sostanza interferente utilizzando il protocollo BS EN 14675:2006 riconosciuto come protocollo standard della Comunit\ue0 Europea. Le analisi effettuate hanno evidenziato una potente attivit\ue0 virucida del prodotto analizzato verso Betanodavirus evidenziando l\u2019assenza di attivit\ue0 virale residua dopo trattamento con Virkon\uaeS e suggerendo una sua possibile applicazione efficace per il controllo dell\u2019ERV nel settore dell\u2019acquacoltura marina

DEVELOPMENT OF AN ABSOLUTE QUANTIFICATION METHOD FOR SEA BASS (DICENTRARCHUS LABRAX) CYTOKINE GENES EXPRESSION

A Real Time PCR was set up for the detection and absolute quantification of a sea bass (Dicentrarchus labrax) cytokine gene panel. To quantify the cytokine expression by real time RT-PCR we used the standard curve method. With this method, quantification results in absolute copy number per l. Specific fragment were selected for 3 cytokine genes: IL-1, IL-6, IL-8 and for S18 of sea bass. For constructing a standard curve we used cDNA plasmid standards. Total RNA was extracted from a tissue that abundantly expressed the target genes, they were amplified by RT-PCR using the same primers as for real time RT-PCR. The amplied targets were purified, ligated in to the il pCR\uae 4-TOPO\uae VECTOR and transformed in to the TOP10 Chemically competent E. coli cells. Finally plasmid DNA was purified and linearized to generate a robust and suitable standard for long-term study purposes. The linearized plasmids were visualized and quantified by agarose gel electrophoresis using a 1 Kbp DNA ladder and the corresponding copy number/l was calculated. The Real Time PCR assay was performed with BRYTTM Green chemistry on the 7300 Real Time PCR System (Applied Biosystem). The BRYTTM Green dye is a DNA-binding molecule with a fluorescence spectrum similar to SYBR Green I. After the amplification cycle a dissociation stage was added to estimated the specificity of the products. To evaluate the limit of detection of the assays 10-fold dilutions of the plasmid were amplified. For each target genes was obtained between 0,3 and 2,4x109 copie/\ub5l of plasmid. Confirmation of the specificity of the amplification reaction was shown by formation of a melting curve with a single peak for each target gene indicating the formation of specific PCR products. The assay allowed analytical enumeration of 24.5, 18, 16 and 3,1 copies/\ub5l of plasmid respectively for IL-1, IL-6, IL-8 and S18. All the analysis returned an R2≥0,99 and an efficiency of 1.00  0.20. Intra-assay variability was calculated for each cytokine and resulted between ≤0,5 and ≤0,9 Std Dev Ct showing a high reproducibility. The method revealed to be very sensitive, reproducible and accurate technique for determining mRNA levels of 3 sea bass cytokines