Coxiella burnetii type IVB secretion system region I genes are expressed early during the infection of host cells.

Analysis of the Coxiella burnetii RSA 493 (Nine Mile phase I strain) genome revealed ORFs with significant homology to the type IVB secretion system (T4BSS) of Legionella pneumophila. The T4BSS genes exist primarily at two loci, designated regions I (RI) and II. In C. burnetii, little is known about the T4BSS regions and the role they play in establishing and/or maintaining infection. Coxiella burnetii T4BSS RI contains genes arranged in three linkage groups: (1) icmW→CBU1651→icmX, (2) icmV→dotA→CBU1647, and (3) icmT→icmS→dotD→dotC→dotB→CBU1646. We used reverse transcriptase (RT)-PCR to demonstrate transcriptional linkage within the groups, and that icmX, icmV, and icmT are transcribed de novo by 8 h post infection (hpi). We then examined the transcript levels for icmX, icmW, icmV, dotA, dotB, and icmT during the first 24 h of an infection using quantitative RT-PCR. The expression initially increased for each gene, followed by a decrease at 24 hpi. Subsequently, we analyzed IcmT protein levels during infection and determined that the expression increases significantly from 8 to 24 hpi and then remains relatively constant. These data demonstrate temporal changes in the RNA of several C. burnetii T4SS RI homologs and the IcmT protein. These changes correspond to early stages of the C. burnetii infectious cycle.

Prophylaxis after exposure to Coxiella burnetii.

Coxiella burnetii is a category B bioterrorism agent. We numerically evaluated the risks and benefits from postexposure prophylaxis (PEP) after an intentional release of C. burnetii to the general population, pregnant women, and other high-risk populations. For each group, we constructed a decision tree to estimate illness and deaths averted by use of PEP/100,000 population. We calculated the threshold points at which the number of PEP-related adverse events was equal to the cases averted. PEP was defined as doxycycline (100 mg 2x/day for 5 days), except for pregnant women, where we assumed a PEP of trimethoprim-sulfamethoxazole (160 mg/800 mg 2x/day) for the duration of the pregnancy. PEP would begin 8-12 days postexposure. On the basis of upper-bound probability estimates of PEP-related adverse events for doxycycline, we concluded that the risk for Q fever illness outweighs the risk for antimicrobial drug-related adverse events when the probability of C. burnetii exposure is >or=7% (pregnant women using trimethoprim-sulfamethoxazole = 16%).

IS1111 insertion sequences of Coxiella burnetii: characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates.

BACKGROUND: Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region approximately 500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method. RESULTS: Isolates Nine Mile phase II, Nine Mile RSA 514, Nine Mile Baca, Scottish, Ohio, Australian QD, Henzerling phase I, Henzerling phase II, M44, KAV, PAV, Q238, Q195 and WAV were tested by PCR and compared to 9Mi/I. Sequencing was used to determine the exact differences in isolates which lacked specific IS elements or produced PCR products of differing size. From this data, an algorithm was created utilizing four primer pairs that allows for differentiation of unknown isolates into five genomic groups. Additional isolates (Priscilla Q177, Idaho Q, Qiyi, Poker Cat, Q229 and Q172) and nine veterinary samples were characterized using the algorithm which resulted in their placement into three distinct genomic groups. CONCLUSION: Through this study significant differences, including missing elements and sequence alterations within and near IS element coding regions, were found between the isolates tested. Further, a method for differentiation of C. burnetii isolates into one of five genomic groups was created. This algorithm may ultimately help to determine the relatedness between known and unknown isolates of C. burnetii.

Strain and phase identification of the U.S. category B agent Coxiella burnetii by matrix assisted laser desorption/ionization time-of-flight mass spectrometry and multivariate pattern recognition.

Accurate bacterial identification is important in diagnosing disease and in microbial forensics. Coxiella burnetii, a highly infective microorganism causative of the human disease Q fever, is now considered a U.S. category B potential bioterrorism agent. We report here an approach for the confirmatory identification of C. burnetii at the strain level which involves the combined use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and supervised pattern recognition via Partial Least Squares-Discriminant Analysis (PLS-DA). C. burnetii isolates investigated in this study included the following prototype strains from different geographical and/or historical origins and with different antigenic properties: Nine Mile I, Australian QD, M44, KAV, PAV, Henzerling, and Ohio. After culture and purification following standard protocols, linear MALDI-TOF mass spectra of pure bacterial cultures were acquired in positive ion mode. Mass spectral data were normalized, baseline-corrected, denoised, binarized and modeled by PLS-DA under crossvalidation conditions. Robustness with respect to uncontrolled variations in the sample preparation and MALDI analysis protocol was assessed by repeating the experiment on five different days spanning a period of 6 months. The method was validated by the prediction of unknown C. burnetii samples in an independent test set with 100% sensitivity and specificity for five out of six strain classes.

Ambient generation of fatty acid methyl ester ions from bacterial whole cells by direct analysis in real time (DART) mass spectrometry.

Direct analysis in real time (DART) is implemented on a time-of-flight (TOF) mass spectrometer, and used for the generation of fatty acid methyl esters (FAMEs) ions from whole bacterial cells.

Analysis of the O-antigen biosynthesis regions of phase II isolates of Coxiella burnetii.

The O-antigen-encoding region in the genomes of 14 isolates of Coxiella burnetii was examined by PCR. Five phase I isolates (Nine Mile clone 7, KAV, Ohio, Henzerling RSA 343, Q173) were analyzed and no deletions were detected. Two other isolates of unknown phase (Scottish, WAV) were examined, but no deletions were detected. In contrast, RSA 514 and three phase II isolates (Nine Mile phase II clone 4, Nine Mile phase II clone 1, Nine Mile Baca) contained large deletions, and the latter two were further characterized by DNA sequencing. Three other phase II isolates (Henzerling RSA 331, M44, Australian QD) contained no apparent deletions. Reactivity to phase I- and phase II-specific antibodies by immunofluorescence assay was used to further characterize isolates. Selected ORFs in Australian QD and M44 DNA were sequenced to detect mutations, and no significant changes were found. Australian QD RNA was examined by reverse transcriptase-PCR specific to the four ORFs hypothesized to encode the O-antigen sugar virenose, which this isolate has been shown to lack, as well as one that is predicted to encode part of the O-antigen ABC transporter. Each of these five genes was found to be expressed.

Rocky Mountain spotted fever, Panama.

We describe a fatal pediatric case of Rocky Mountain spotted fever in Panama, the first, to our knowledge, since the 1950s. Diagnosis was established by immunohistochemistry, PCR, and isolation of Rickettsia rickettsii from postmortem tissues. Molecular typing demonstrated strong relatedness of the isolate to strains of R. rickettsii from Central and South America.

National surveillance and the epidemiology of human Q fever in the United States, 1978-2004.

Although Q fever is considered enzootic in the United States, surveillance for human Q fever has been historically limited. From 1978 through 1999, 436 cases (average = 20 per year) of human Q fever were reported. After Q fever became nationally reportable in 1999, 255 human Q fever cases (average = 51 per year) were reported with illness onset during 2000 through 2004. The median age of cases was 51 years, and most cases were male (77%). The average annual incidence of Q fever was 0.28 cases per million persons, and was highest in persons 50-59 years of age (0.39 cases per million). State-specific incidence ranged from a high of 2.40 cases per million persons in Wyoming, to 0 cases in some states. Since Q fever became reportable, case reports have increased by more than 250%. Surveillance for Q fever is essential to establish the distribution and magnitude of disease and to complement U.S. bioterrorism preparedness activities.

Prevalence of antibodies to Coxiella burnetii among veterinary school dairy herds in the United States, 2003.

Prevalence of antibodies to Coxiella burnetii in 24 veterinary school-associated dairy herds in the United States was assessed through laboratory testing of bulk tank milk specimens by indirect immunofluorescent antibody assay. Twenty-two herds (92%) had evidence of antibodies to C. burnetii Phase I antibodies at a titer of > or = 1:16, and nine herds (38%) had Phase I antibody titers of > or = 1:256. These results suggest that C. burnetii infection is geographically widespread among dairy herds in the United States.

Identification of biomarkers of whole Coxiella burnetii phase I by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) specific biomarkers have been shown to be an effective tool for identifying microorganisms. In this study, we demonstrate the feasibility of using this technique to detect the obligate intracellular bacterium Coxiella burnetii, a category B bioterrorism agent. Specific biomarkers were detected in C. burnetii Nine Mile phase I (NMI) strain purified from embryonated egg yolk sac preparations. Whole organisms were applied directly to the MALDI target. MALDI-TOF MS analysis of C. burnetii NMI grown and purified at different times and places revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 1000-25000 Da. Statistical analysis of the averaged centroided masses uncovered at least 24 peptides or biomarkers. Three biomarkers observed in the MALDI-TOF MS spectrum consistently matched proteins that had been previously described in C. burnetii, one of them being the small cell variant protein A. MALDI-TOF MS analysis of whole organisms represents a sensitive and specific option for characterizing C. burnetii isolates, especially when coupled with antigen capture techniques. The method also has potential for several applications in basic microbial research, including regulation of gene expression.

Complete genome sequence of the Q-fever pathogen Coxiella burnetii.

The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies of C. burnetii resemble that of Rickettsiae and Chlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in the C. burnetii genome. These analyses suggest that the obligate intracellular lifestyle of C. burnetii may be a relatively recent innovation.

Permeability of Coxiella burnetii to ribonucleosides.

Knowledge about transport in Coxiella burnetii, an obligate phagolysosomal parasite, is incomplete. The authors investigated the capability of isolated, intact, host-free Coxiella to transport ribonucleosides while incubated at a pH value typical of lysosomes. Because of the low activities and limitations of obtaining experimental quantities of isolated, purified Coxiella, incorporation of substrate into nucleic acid was used as a trap for determination of uptake abilities. Virulent wild-type (phase I) organisms possessed uptake capability for all ribonucleosides. Both phase I and phase II (avirulent) organisms incorporated the purine nucleosides guanosine, adenosine and inosine, and showed a more limited uptake of thymidine and uridine. Both phases were poorly active in cytidine uptake. Neither phase of the organism was capable of transport and incorporation of NTPs, CMP, cytosine or uracil. Water space experiments confirmed that the uptake process concentrated the purine nucleosides within the cytoplasm of both wild-type and phase II Coxiella via a low-pH-dependent mechanism. Comparison of uptake rates in Escherichia coli versus Coxiella verified that the incorporation of ribonucleosides by Coxiella is a slow process. It is concluded that Coxiella possesses some transport pathways consistent with utilization of pools of nucleosides found within its host cell lysosomal pathway.