The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-m pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm 2 ) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% ؎ 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation.Legionella pneumophila is widespread in natural freshwater environments, despite its fastidious nature (16). The bacterium has also frequently been observed in engineered water systems such as warm water distributing systems, cooling towers, humidifiers, and fountains (25,43,50). The multiplication of the organism in water systems poses a potential human health risk when aerosolization can occur (12).Legionellae grow in vitro only in complex media with supplements of cysteine and iron salts (11). For their multiplication in vivo, other microorganisms are required, but different species of heterotrophic bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, and Flavobacterium sp.) alone do not support the growth of L. pneumophila (31,45). In vitro studies using cocultures have repeatedly demonstrated the intracellular multiplication of L. pneumophila in amoebae (Acanthamoeba, Echinamoeba, Hartmannella, Naegleria, Vahlkampfia, and Dictyostelium) and in a ciliated protozoon (Tetrahymena pyriformis) (12,15,21,39). Amoebae have been observed in water systems associated with Legionnaires' disease (5, 7), and L. pneumophila can recolonize water distributing systems within a few weeks after disinfection (26,27). Batch experiments with tap wate...

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 ؋ 10 ؊1 and 1.14 ؋ 10 4 cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 ؎ 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (>98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

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Field margin management is a common measure employed in Europe to support farmland bird populations. In this study we found and analysed 237 nests of the Skylark Alauda arvensis in the Netherlands over a period of 6 years to determine the effects of arable field margins and breeding crop on nest-level reproductive success. Additionally, the effect of field margins on predation was investigated and food availability in crops and field margins was compared. Neither clutch size, nest survival nor nestling body weight were improved by field margin availability, irrespective of the breeding crop used. However, the choice of breeding crop had important effects. Nestling weight was significantly lower in cereals than in grassland and lucerne, corresponding with the low prey densities present in cereals. Nest survival was lowest in grassland due to frequent silage cutting. Predation rates were highest in cereals but were not affected by field margin proximity. The highest reproductive success was achieved in lucerne, which was mown twice a year and retained a suitable height for breeding throughout the breeding season. We conclude that field margins are not sufficient to maintain a Skylark population in this intensively farmed area. The presumably more subtle effects of increased food availability cannot compensate for the high nest failure rates resulting from agricultural operations and predation. In this and similar areas, the provisioning of safe nesting habitat throughout the breeding season is essential to improve breeding performance. Our research suggests that this can be achieved by reducing the frequency of silage cutting on grassland and by increasing the surface area of lucerne.