The world's largest Q fever outbreak is ongoing in The Netherlands with around 3,000 confirmed cases since the first half of 2007. Increased awareness has resulted in early referral of patients for diagnostics. An important drawback to serological diagnosis of acute Q fever is the lag phase in antibody response. Therefore, we evaluated the performance of a real-time PCR for detection of Coxiella burnetii DNA using serum samples from patients with acute Q fever. PCR, targeting IS1111, was retrospectively performed on acute-phase and follow-up convalescent-phase serum samples from 65 patients with acute Q fever as diagnosed by immunofluorescence assay. The results obtained by PCR were related to disease stage as defined by subsequent appearance of phase II IgM, phase II IgG, phase I IgM, and phase I IgG (IgM-II, IgG-II, IgM-I, and IgG-I, respectively) antibodies and time since onset of disease. In addition, we analyzed seronegative acute-phase serum samples from patients with inconclusive Q fever serology, because no convalescent-phase serum samples were available. PCR was scored positive in 49/50 (98%) seronegative sera, 9/10 (90%) sera with isolated IgM-II antibodies, 3/13 (23%) sera with IgM-II/IgG-II antibodies, 2/41 (5%) sera with IgM-II/IgG-II/IgM-I antibodies, 0/15 (0%) sera with IgM-II/IgG-II/IgM-I/IgG-I antibodies, and 0/1 (0%) serum sample with IgM-II/IgG-II/IgG-I antibodies. The latest time point after onset of disease in which C. burnetii DNA could be detected was at day 17. In patients with inconclusive Q fever serology, PCR was positive in 5/50 (10%) cases. We conclude that real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever. C. burnetii DNA becomes undetectable in serum as the serological response develops.

We now know that the abundance of free viruses in most marine environments is high. There is still, however, a lack of understanding of their occurrence and distribution and of in situ relationships between viral and host communities in natural environments. This may be partly due to methodological limitations. Our main aim was therefore to perform a case study in which a variety of methods were applied in order to give an improved, high-resolution description of the microbial communities in a natural environment. In order to do this we combined light microscopy (LM), transmission electron microscopy (TEM), flow cytometry (FCM), PCR denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) and studied the diversity and succession of algae, bacteria and viruses in a nutrient enriched seawater enclosure. In the enclosure we experienced a situation where the development of the dominating algal population, which consisted of several flagellate species, was followed by proliferation of several different size-classes of viruses. The total bacterial number decreased markedly during the flagellate bloom but the community composition was maintained and the diversity remained high. Our results indicate a close linkage between various algal, bacterial and viral populations and show that virioplankton do not necessarily terminate algal and bacterial blooms but that they keep the host populations at non-blooming levels.KEY WORDS: Bacteria · DGGE · Diversity · Flow cytometry · Light microscopy · PFGE · Phytoplankton · VirusResale or republication not permitted without written consent of the publisher

During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities.

Several previous studies have shown that Emiliania huxleyi blooms and terminations have been succeeded by an increase in large virus-like particles (LVLP), strongly suggesting the bloom collapse was caused by viral lysis. However, due to methodological limitations, knowledge of how such blooms affect the rest of the microbial community is limited. In the current study we induced a bloom of E. huxleyi in seawater enclosures and applied methods enabling us to describe the algae, bacteria and virus communities with greater resolution than has been done previously. The development of the dominating algal, viral and bacterial populations in the nutrient-amended seawater enclosures was followed by flow cytometry (FCM). Light microscopy (LM), PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pulsed-field gel electrophoresis (PFGE) were used to describe the changes in community composition in greater detail. The algal community was dominated by E. huxleyi until termination of the bloom by viral lysis. After bloom termination the additional algal populations present in the enclosures increased in abundance. A marked increase in viruses other than the one infecting E. huxleyi was also observed. Total bacterial number and community composition were also greatly influenced by the bloom and its collapse. KEY WORDS: Diversity · Emiliania huxleyi · Microbial community · Viral lysisResale or republication not permitted without written consent of the publisher