Chlorine inactivation of coxsackievirus B5 in recycled water destined for non-potable reuse.

Currently guidelines for disinfection of water with free chlorine, while primarily developed for potable water, are often used for virus disinfection of nitrified recycled water of >1 NTU (Nephelometric Turbidity Unit). More information is needed on the disinfection efficacy of free chlorine for viruses in waters of varying turbidity and pH due to significant reuse of treated wastewater of varying quality. In this study, disinfection efficacy in nitrified/denitrified activated sludge treated wastewater was investigated for coxsackievirus B5 (CB5), an enterovirus known to be highly resistant to free chlorine. The required chlorine contact times (CT) values (mg.min/L) for inactivation of CB5 were established in treated wastewater at 10 °C and of varying turbidity (0.2, 2, 5 and 20 NTU) and pH (7, 8 and 9). CTs were calculated to achieve 1 to 4 log10 inactivation. Robust data is presented in support of the chlorine CT values required to inactivate a chlorine-resistant virus in a range of turbidities and pHs in treated wastewaters. The testing method used a conservative approach and the data presented have been used to develop the free chlorine virus inactivation guildelines for recycled water in Victoria and South Australia, Australia.

Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments.

Characterisation of the protists of cold environments provides important background for assessing the effects of climate change on microbial communities. Tetramitus angularis n. sp., from aquatic environments in Iceland and Switzerland, is the first vahlkampfiid recognised to have a characteristic Tetramitus flagellate stage combined with pre-formed excystment pores, which are not typical of this genus. T. angularis amoebae have a typical vahlkampfiid locomotive form and contain prominent lipid inclusions. Flagellates have a collar and cytostome, and can be mono- to multi-nucleate with corresponding change in cell shape from cylindrical to ellipsoidal and variable number of flagella. Cysts are round to semi-angular and have 2-5 pores closed by protruding, translucent plugs. A second organism, T. parangularis n. sp. from Alaska, has similar cysts but a flagellate stage has not been recognised; ITS sequence divergence is consistent with species criteria in the Vahlkampfiidae. Phylogenetic analysis of sequence data for the 5.8S rDNA region clusters the new spp. with T. rostratus, T. entericus and T. waccamawensis.

Detection and significance of the potentially pathogenic amoeboflagellate Naegleria italica in Australia.

Thermophilic amoeboflagellates in the genus Naegleria include both virulent and benign species. One of the less studied species, N. italica, has not been detected in the environment since the first reports from Italy in the 1980s; its virulence is known only from infection of laboratory mice. Two recent strains from recreational water in Western Australia (AWQC NG960, NG961) were tentatively identified as N. italica from the characteristic mobilities of seven isozymes. Sequences of the 5.8S rRNA gene and its flanking ITS aligned with a 380+bp length of the published sequence for N. italica with 98% identity. Differences from the type strain were confined to ITS2. Shorter alignments (<320 bp) were observed with other Naegleria species, corresponding to conserved regions of the 5.8S gene and ITS. Unlike the European type strain of N. italica, the Australian isolates failed to infect laboratory mice intranasally, confirming that infectivity of this species is variable and often lower than in N. fowleri.

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis.

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Density and diversity of protozoa in some arid Australian soils.

This is the first extensive study of soil protozoa of arid lands. Twenty-six samples from litters, soils, termitaria, and a cyanobacterial crust, collected from central and south Australian arid lands, were analyzed for numbers and species of gymnamoebae, ciliates, and testacea. Amoebae ranged from 1,000-5,000/g of material, and were two orders of magnitude more abundant than ciliates. Both groups increased in abundance and species richness from bare soils through spinifex to mulga to chenopod vegetations. Testacea ranged 900-5,000/g with similar species richness throughout vegetations, but reached 11,900/g with a doubling of species in a refugium in Kings Canyon. The most prevalent species of amoebae, ciliates, and testacea were taxa associated with ephemeral and disturbed habitats (r-selection). The cyanobacterial crust might be considered a micro-refugium because it contained a number of non-encysting protozoa, including Thecamoeba sp. and Nassula picta, feeding on cyanobacterial filaments. The numbers and species richness of protozoa under shrubs were greater than in bare soils, supporting the resource island hypothesis that desert plants create soil heterogeneity by localizing soil fertility under their canopies.