Laura Volterra

Molecular techniques for the identification of enteric viruses in marine waters

Abstract The RNAs extracted from 28 high spin pellets of clarified lysates from BGM cells, previously infected by concentrated Adriatic Sea water samples, were analyzed by a dot-blot test. We used a 32 P-labeled cDNA probe ( BamH I 220–670 ) from the 5′ non-coding end of the cloned poliovirus I (Mahoney) genome. The probe was selected for its broad range of genetic specificities. Based on the density of the hybridization signals on the autoradiograph, our dot-blot results showed a high degree of reactivity of the probe to homologous (poliovirus 1) and heterologous (echovirus 6 and coxsackievirus B3) reference RNA, as predicted by a computer analysis of their sequences, and a low reactivity to that of the samples leading us to exclude the presence of enteroviruses like the reference strains. In order to understand if there were other enteroviral serotypes or not, dot-blot tests were supplemented with Northern-blot hybridization assays. Results from the Northern-blot showed a series of fragments ranging from 4.3 to 1.2 kb but no signal corresponding to 7.5 kb (as did the positive control RNAs). These features suggested that the tested RNAs might derive from viruses of the Reoviridae family, which includes members with segmented genome. Traditional PAGE analysis showed clearly the 10-fragments pattern characteristic of reoviruses. Twenty-three out of the twenty-eight samples tested showed the presence of viruses, and this confirms the previously noted cytopathic effect (CPE) of the samples on the BGM cells.

Biofilm amount estimation by fluorescein diacetate

Various methods for direct and indirect biofilm amount estimation are available but most of them have been developed on free cell cultures and/or their application to biofilm analysis often implies biofilm removal from the solid surface or extraction procedures. This work presents a method to measure biofilm bacterial activity, that does not require the removal step. It uses fluorescein diacetate (FDA), a fluorogen (fluorescein) conjugated to two acetate radicals. This compound, once hydrolyzed by an exoenzyme that is present in almost all bacteria, releases the fluorogen that has an absorbance at 490 nm. Experimental tests have shown a good correlation between the absorbance of the solution at 490 nm and bacterial concentrations. This method may be particularly helpful in potable water pipe control to indicate where and when it is necessary to perform corrective actions to stop and/or reduce biofilm growth.

Microcystin-like toxins in different freshwater species of Oscillatoria

In January and September of 1989 and March 1990 blooms of Oscillatoria rubescens, Oscillatoria tenuis and Oscillatoria mougetii were found in Lake Simbirizzi and Lake Flumendosa in Sardinia, and in Lake San Puoto in the Lazio region of Italy. By using different extraction methods and HPLC analysis, two microcystin-like toxins (RR-like and YR-like), similar to some of the toxic compounds produced by the Cyanophycea Microcystis aeruginosa, were detected in these blooms.

Some features of a bloom of Oscillatoria rubescens D.C. registered in two italian reservoirs

This paper summarizes biological, microbiological, physico-chemical and toxicological analyses carried out during a bloom of Oscillatoria rubescens D.C. in two Sardinian lakes that began in January and lasted 7 mo. The algal species was found to be biotoxins producer. The toxicity was confirmed through laboratory tests. Acute LD50 in mice was about 120 mg kg−1 body weight i.p. and at the necroscopic examination animals showed ochraccous dashes on the liver. When subacute p.o. toxicity tests were performed, in liver cells swollen, fragmented or twinned nuclei appeared in a dose related manner. During the bloom the eutrophic lakes exhibited a reduced number of microbial fecal indicators and a rich population of environmental bacterial flora.

Chemical composition and biological origin of ‘dirty sea’ mucilages

Abstract The carbohydrate composition of the extracellular exudates of the native algae cells floating in the Adriatic sea during the summer have been determined. These results were then compared with similar analyses performed on the excreted organic matter produced by in vitro cultures of Amphora coffeaeformis and Cylindrotheca fusiformis , both of which have been isolated in the past in the same areas of the Adriatic sea. Separation of the jelly-like polymeric components of the mucilages is described. The insoluble fraction presents a monomer distribution similar to that of C. fusiformis , while the unprecipitable fraction is similar to that of A. coffeaeformis . Native mucilages appear to be an intimate mixture of the extracellular exudates produced by the in vivo metabolism of the two diatoms.

Marine snow in the Adriatic Sea : a multifactorial study

Chemical characterization (elementary composition, carbohydrate and protein content) as well as biological determination (presence of phytoplankton, chlorophyll a and pheopigment content) have been carried out on two samples of gelatinous aggregates. On the basis of the results obtained and of general meteorological and hydrodynamic conditions recorded in 1988 and 1989, a hypothesis of gelatin formation in proposed

Coliphages as indicators in a treatment plant

The potential use of coliphages as indicators of water pollution was investigated. Several strains ofE. coli hosts were used in order to detect the better marker. Analyses were carried out on a treatment plant for the convenience of phages recoveries. Results show the possibility that strains different fromE. coli 9484B can show better recoveries of PFU m1−1.

Fecal streptococci recoveries in different marine areas

The usual media and procedures were followed to measure the concentration of fecal streptococci (MPN on Azide Dextrose and Ethyl Violet Azide broths, membrane filtration on m-Enterococcus, KF and Pfizer Selective Enterococcus agars and according to the mE procedure) in samples collected along two different marine areas. The results were evaluated on the basis of three parameters: total concentrations, number of enterococci-like colonies (namely colonies gram positive, catalase negative, coccus shaped) and rate of strictly named fecal streptococci.From the results it appears that the various media and procedures employed gave different yields and their capacity to measure fecal streptococci varies according to the origin of samples. The accompanying bacterial flora may play an important role on the selectivity of each technique to measure the fecal streptococci.

Enteric virus pollution of Tyrrhenian areas

Abstract63 samples (53 seawater and 10 estuarine water samples) of 20 L were obtained during a bathing season from 47 seawater stations and from 1 estuarine station. To determine viral pollution, all samples were subjected to two different methods of viral concentration: tangential ultrafiltration and adsorption-elution with electropositive membranes. Detection of viruses was by cytopathic effect (CPE) in BGM and NA 104 cells. Isolates were identified by dot-blot hybridization and Polyacrylamide Gel Electrophoresis (PAGE). While estuarine water showed enterovirus and/or reovirus presence in 100% of samples, only 14 stations of 47 seawater station samples (30%) showed viral contamination: enteroviruses were isolated from 6 and reoviruses from 8 of the 14 stations. 28 unidentified viruses were detected from seawater in MA104 cells by CPE whereas these viruses were not detected in BGM cells. Enterovirus recovery seems to be better when water samples are concentrated by the tangential ultrafiltration than with absorption-elution with electropositive membranes. For reoviruses and the other viruses the two methods were almost equivalent. BGM cells seem to be more susceptible to enteroviruses, MA104 to reoviruses. Reoviruses failed to indicate enterovirus presence as most of enteroviruses were isolated in waters where reovirus was not observed. Isolated viral species distribution changed during bathing season.

Diarrhoeic shellfish toxins in Adriatic Sea mussels evaluated by an ELISA method

A competitive enzyme immunoassay (ELISA) was used to determine the presence of okadaic acid (OA) and/or dinophysistoxin-1 (DTX-1) in mussels. The accuracy and sensitivity of the ELISA method has been checked. The sensitivity of the method (100 ng/g of hepatopancreas) makes it possible to determine OA concentrations ten times lower than the tolerance limits established by the Health Authorities of many countries. For the first time, OA and/or DTX-1 were detected in mussels (Mytilus galloprovincialis) collected in different stations along the Adriatic Sea coasts.