L Cantiani

Assessing airborne biological hazard from urban wastewater treatment

The production of microbial aerosols by urban sewage treatment plants may have wide hygienic implications which call for careful evaluation: exposure to such aerosols may in fact represent a health hazard for plant workers and nearby residents alike. This paper describes the results of a study analysing the degree of microbial contamination at diAerent levels of treatment at several plants in the City of Leghorn (Livorno, Italy). Monthly aerosol samples were collected with an agar impact sampler from January to November 1996, from diAerent sites at an activated sludge plant, an anaerobic sludge plant and a wastewater washing station. The total bacterial and coliform counts were determined, and pathogenic enteric bacteria and viruses were determined. These same parameters were also measured in wastewater and sludge samples obtained at the same sites. The results revealed that high-grade airborne contamination existed at several of the studied sites. In particular, pathogenic enteric bacteria (Salmonella enteritidis and S. boydii) were isolated in 2% of the samples (540 l per sample), reovirus in 46% and enterovirus in 9% (1800 litres per sample in indoor environment and 3000 l outdoor), always in association with the former virus. The biological parameters measured had no evident correlation with meteorological factors such as temperature, relative humidity or wind characteristics. Viral contamination proved to be quite wide-spread and detectable even in the presence of low levels of bacterial contamination. Although virological analysis have been only qualitative, and the diAerent volumes examined for viruses and bacteria cannot allow us to appraise with accuracy the association between these two parameters, the viral presence along with low bacteria contamination suggests more dedicated studies to address with greater accuracy the quantitative aspects of this association. However the monitoring performed allowed for a determination of the areas of greatest potential risk for plant workers, and the preventive measures most suitable to guaranteeing their safety. # 2000 Elsevier Science Ltd. All rights reserved

Enteric virus detection in Adriatic seawater by cell culture, polymerase chain reaction and polyacrylamide gel electrophoresis

Abstract Forty samples of sea and estuary water were collected from a 40 km strip along the Adriatic coast of Italy between June 1994 and September 1995. Each sample consisted of 10 l of water. Routine bacteriological analyses were carried out and viral particles concentrated on cross-flow membranes; the concentrated water samples, equally divided into two parts, were used to infect both BGM and Hep-2 cells. Lysates from all cell cultures were further tested for the presence of enteroviruses by reverse-transcribed polymerase chain reaction (RT-PCR) and reoviruses by polyacrylamide gel electrophoresis (PAGE). The results showed widespread viral contamination of the waters tested, particularly in late summer. Under our experimental conditions, BGM cells were more efficient than Hep-2 in recovering viruses. In fact, enteroviruses were detected in up to 33% and reoviruses in 80% of BGM infected with seawater, compared to 8% and 53%, respectively, for the Hep-2 cells. In estuarine samples, enteroviruses were detected in 30% and reovirus in 54% of BGM, compared to 23% and 30% of Hep-2. Twenty nine out of 40 samples showed the presence of infectious particles on the basis of the CPE appearance; after identification of the isolated viruses, only 13 turned out to be specifically contaminated by enteroviruses. Of the latter, five were below the bacteriological standards set by the Italian legislation in line with the EEC Directive 76/160 IEEC for bathing waters.

A new RT-PCR method for the identification of reoviruses in seawater samples

The frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. In order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (RT-PCR) was set up to detect reoviruses. Two primers were engineered to amplify a 538 base pair fragment of the Sigma 2 gene. Reovirus strains obtained from ATCC (Jones, Lang, Dearing, Abney, NC-TEV, SV59 and SV12) were used as references. Twenty-four samples of 101 were collected from two beaches of the Adriatic sea and 12 from the neighbourhood of Fano Harbour Channel. The presence of environmental reoviruses was tested on both concentrated seawater samples and lysates of BGM cells infected with the concentrated seawater samples. The new method was used in parallel with the detection of a 3:3:4 electrophoretic pattern of reovirus RNA in polyacrylamide gel electrophoresis (PAGE). Enterovirus and bacteria were also screened in compliance with EEC directives. No enteroviruses were isolated, and it was not attributable to reovirus interference. All the reovirus found by PAGE (8/72) were confirmed by RT-PCR, while several genomes (14/72) were detected only by RT-PCR. Presumptive methods of virus identification, that is CPE on BGM cells and haemagglutination test, were not able to detect them. The specificity of RT-PCR products was checked by direct nucleotide sequence analyses of the amplicons. The phylogenetic analyses showed heterogeneous taxa including human and animal reoviruses, with strong evidence that they were spreading consistently from the Harbour-Channel. This novel approach for reovirus detection will be very useful as a trace route of faecal pollution; more importantly, it could be very useful in contributing to the creation of a databank of circulating enteric viruses.

Molecular analysis of poliovirus 3 isolated from an aerosol generated by a waste water treatment plant

Abstract We examined three samples of lysates from cell cultures that had previously been infected by the aerosol generated by a waste water treatment plant near Pisa. We first attempted to confirm that we were dealing with one of the enterovirus family, using an inverse PCR analysis of the RNA extracted from the cell lysates. This identified a single genome sequences in all the samples, which corresponded to poliovirus 3. Sequence analyses revealed that the genotyping of poliovirus 3 was accurate for the species, irrespective of the genomic region sequenced. Subspecies genotyping is only possible for the translated region, and in this case, it identified the Poliovirus type 3 Leon 12a1b strain as ancestor of the virus isolated. Molecular analyses were then carried out on the wild virus in the regions VP2 (9623 bp), VP3 (922 bp) and VP1 (1128 bp) and found various nucleotide mutations (e.g. 472 T→C ; 970 G→C, Cys→Ser ; 1319 G→T, Val→Leu ; 1743 T→C, Val→Ala ; 1817 C→T, Hys→Tyr ). Thermosensitivity tests at 34°C and 44°C showed a slight reversion to the heat resistant phenotype. The need for adequate protective measures for plant staff, together with the frequent use of treated water for irrigation, means that effective methods of aerosol borne pathogen detection and risk determination must be adopted.