Genome analysis and heterologous expression of acetate-activating enzymes in the anammox bacterium Kuenenia stuttgartiensis.

Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.

Evaluation of a diagnostic algorithm for acute Q fever in an outbreak setting.

In the peak of the 2009 Q fever outbreak in the Netherlands, we introduced a diagnostic algorithm for acute Q fever with an enzyme-linked immunosorbent assay for immunoglobulin M antibodies to Coxiella burnetii phase II antigens (MII screen) as an initial step. Subsequently, an immunofluorescence assay or PCR was performed depending on the MII screen outcome, date of onset of disease, and inpatient or outpatient setting. The impact of MII screen on the number of immunofluorescence assays performed and the contribution of PCR to diagnosis were retrospectively evaluated in 825 patients referred in a 17-day period. Acute Q fever was diagnosed in 256 patients. The introduction of MII screen reduced the number of immunofluorescence assays performed by more than 80%. In 103 patients, PCR analysis contributed to the diagnosis of acute Q fever. Q fever diagnostics were hampered by the fact that for a high number of patients the date of onset of disease was not provided and the requested follow-up serum samples were not received.

Coxiella burnetii DNA in goat milk after vaccination with Coxevac(®).

Q fever is a zoonotic disease caused by Coxiella burnetii, a species of bacteria that is distributed globally. A large Q fever epidemic is currently spreading throughout the Netherlands with more than 3500 human cases notified from 2007 to 2009. Governmental measures to prevent further spread of the disease imposed in December 2009 included vaccination of all dairy goats and sheep and, in parallel, bulk tank milk testing to identify contaminated goat and sheep farms. When bulk tank milk was found to contain C. burnetii DNA, pregnant ruminants were culled. An important, but unsolved issue in this policy was whether vaccine-derived C. burnetii DNA is excreted in milk after vaccination. Using real time PCR and single nucleotide polymorphism (SNP) genotyping techniques, we show here that within hours and up to 9 days after vaccination with Coxevac(®), vaccine-derived C. burnetii DNA can be detected in the milk of dairy goats. This is the first report describing DNAlactia of vaccine-derived DNA after vaccination with a completely inactivated vaccine. This finding had implications for the Dutch policy to combat the Q fever epidemic. A 2-week interval was introduced between vaccination and bulk tank milk testing to identify infected farms.

Single-nucleotide-polymorphism genotyping of Coxiella burnetii during a Q fever outbreak in The Netherlands.

Coxiella burnetii is the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of the C. burnetii strain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination of C. burnetii strains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 different C. burnetii genotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 different C. burnetii genotypes are involved in the Dutch outbreak.

Real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever.

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.