scholarly journals Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

2011 ◽  
Vol 77 (18) ◽  
pp. 6516-6523 ◽  
Author(s):  
A. de Bruin ◽  
A. de Groot ◽  
L. de Heer ◽  
J. Bok ◽  
P. R. Wielinga ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
The Netherlands ◽  
Quantitative Pcr ◽  
Coxiella Burnetii ◽  
Internal Control ◽  
Q Fever ◽  
Qpcr Assay ◽  
Environmental Matrices ◽  
Content Type ◽  
Control Target

ABSTRACTQ fever, caused byCoxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection ofC. burnetiiDNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects threeC. burnetiitargets (icd,com1, and IS1111) and oneBacillus thuringiensisinternal control target (cry1b).Bacillus thuringiensisspores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targetsicd,com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening forC. burnetiiDNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels ofC. burnetiiDNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10- or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both theC. burnetiitargets and the internal control. The inclusion of an internal control target and threeC. burnetiitargets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence ofC. burnetiiDNA during an outbreak.

scholarly journals Detection of Coxiella burnetii DNA on Small-Ruminant Farms during a Q Fever Outbreak in the Netherlands

2012 ◽  
Vol 78 (6) ◽  
pp. 1652-1657 ◽  
Author(s):  
A. de Bruin ◽  
R. Q. J. van der Plaats ◽  
L. de Heer ◽  
R. Paauwe ◽  
B. Schimmer ◽  
...  
Keyword(s):  
The Netherlands ◽  
Surface Area ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Monitoring Program ◽  
Primary Source ◽  
Dairy Goat ◽  
Environmental Matrices ◽  
Content Type ◽  
Bulk Milk

ABSTRACTDuring large Q fever outbreaks in the Netherlands between 2007 and 2010, dairy goat farms were implicated as the primary source of human Q fever. The transmission ofCoxiella burnetiito humans is thought to occur primarily via aerosols, although available data onC. burnetiiin aerosols and other environmental matrices are limited. During the outbreak of 2009, 19 dairy goat farms and one dairy sheep farm were selected nationwide to investigate the presence ofC. burnetiiDNA in vaginal swabs, manure, surface area swabs, milk unit filters, and aerosols. Four of these farms had a positive status during theCoxiella burnetiibulk milk monitoring program in 2009 and additionally reported abortion waves in 2008 or 2009. Eleven farms were reported as having positive bulk milk only, and five selected (control) farms had a bulk milk-negative status in 2009 and no reported Q fever history. Screening by quantitative PCR (qPCR) revealed that on farms with a history of abortions related toC. burnetiiand, to a lesser extent, on farms positive by bulk milk monitoring, generally higher proportions of positive samples and higher levels ofC. burnetiiDNA within positive samples were observed than on the control farms. The relatively high levels ofC. burnetiiDNA in surface area swabs and aerosols sampled in stables of bulk milk-positive farms, including farms with a Q fever-related abortion history, support the hypothesis that these farms can pose a risk for the transmission ofC. burnetiito humans.

scholarly journals Molecular typing of Coxiella burnetii from animal and environmental matrices during Q fever epidemics in the Netherlands

2012 ◽  
Vol 8 (1) ◽  
pp. 165 ◽  
Author(s):  
Arnout de Bruin ◽  
Pleunie TW van Alphen ◽  
Rozemarijn QJ van der Plaats ◽  
Lianne ND de Heer ◽  
Chantal BEM Reusken ◽  
...  
Keyword(s):  
The Netherlands ◽  
Coxiella Burnetii ◽  
Molecular Typing ◽  
Q Fever ◽  
Environmental Matrices

scholarly journals Presence and Persistence of Coxiella burnetii in the Environments of Goat Farms Associated with a Q Fever Outbreak

2013 ◽  
Vol 79 (5) ◽  
pp. 1697-1703 ◽  
Author(s):  
Gilbert J. Kersh ◽  
Kelly A. Fitzpatrick ◽  
Joshua S. Self ◽  
Rachael A. Priestley ◽  
Aubree J. Kelly ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Quantitative Pcr ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Human Movement ◽  
Zoonotic Disease ◽  
Content Type ◽  
Air Samples ◽  
Ruminant Livestock

ABSTRACTQ fever is a zoonotic disease caused by inhalation of the bacteriumCoxiella burnetii. Ruminant livestock are common reservoirs forC. burnetii, and bacteria present in aerosols derived from the waste of infected animals can infect humans. The significance of infection from material deposited in the environment versus transmission directly from infected animals is not known. In 2011, an outbreak of Q fever cases on farms in Washington and Montana was associated with infected goats. A study was undertaken to investigate the quantity and spatial distribution ofC. burnetiiin the environment of these goat farms. Soil, vacuum, and sponge samples collected on seven farms epidemiologically linked to the outbreak were tested for the presence ofC. burnetiiDNA by quantitative PCR. Overall, 70.1% of the samples were positive forC. burnetii. All farms had positive samples, but the quantity ofC. burnetiivaried widely between samples and between farms. High quantities ofC. burnetiiDNA were in goat housing/birthing areas, and only small quantities were found in samples collected more than 50 m from these areas. Follow-up sampling at one of the farms 1 year after the outbreak found small quantities ofC. burnetiiDNA in air samples and large quantities ofC. burnetiipersisting in soil and vacuum samples. The results suggest that the highest concentrations of environmentalC. burnetiiare found in goat birthing areas and that contamination of other areas is mostly associated with human movement.

scholarly journals Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages

2012 ◽  
Vol 80 (6) ◽  
pp. 1980-1986 ◽  
Author(s):  
Laura J. MacDonald ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Coxiella Burnetii ◽  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Bacterial Agent ◽  
Dependent Protein

ABSTRACTCoxiella burnetiiis the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission,C. burnetiireplicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms ofC. burnetiiintracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling duringC. burnetiiinfection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV andC. burnetiireplication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulentC. burnetiiisolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated duringC. burnetiiinfection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA inC. burnetiiinfection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.

scholarly journals Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

2015 ◽  
Vol 83 (3) ◽  
pp. 1190-1198 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Uma M. Sharma ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Secretion System ◽  
Terminal Region ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Content Type ◽  
Type Iv

Coxiella burnetiicauses human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages,C. burnetiiuses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100C. burnetiiDot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in allC. burnetiiisolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizingproteinA). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function duringC. burnetiiinfection.

scholarly journals Distribution of Bacillus thuringiensis subsp. israelensis in Soil of a Swiss Wetland Reserve after 22 Years of Mosquito Control

2011 ◽  
Vol 77 (11) ◽  
pp. 3663-3668 ◽  
Author(s):  
Valeria Guidi ◽  
Nicola Patocchi ◽  
Peter Lüthy ◽  
Mauro Tonolla
Keyword(s):  
Spatial Distribution ◽  
Bacillus Thuringiensis ◽  
Confidence Interval ◽  
Quantitative Pcr ◽  
Mosquito Control ◽  
Soil Samples ◽  
Sampling Point ◽  
Content Type ◽  
Large Numbers ◽  
Continuous Accumulation

ABSTRACTRecurrent treatments withBacillus thuringiensissubsp.israelensisare required to control the floodwater mosquitoAedes vexansthat breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of restingB. thuringiensissubsp.israelensisspores in the soil was measured. TheB. thuringiensissubsp.israelensisconcentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for theB. thuringiensissubsp.israelensis cry4Aaandcry4Bagenes.B. thuringiensissubsp.israelensisspores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number ofB. thuringiensissubsp.israelensistreatments, the elevation of the sampling point, and the duration of the flooding periods. The number ofB. thuringiensissubsp.israelensistreatments was the major factor influencing the distribution of spores in the different topographic zones (P< 0.0001). These findings indicated thatB. thuringiensissubsp.israelensisspores are rather immobile after their introduction into the environment.

scholarly journals Selective Inhibition of Coxiella burnetii Replication by the Steroid Hormone Progesterone

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Zachary P. Howard ◽  
Anders Omsland
Keyword(s):  
Coxiella Burnetii ◽  
Efflux Pump ◽  
Q Fever ◽  
Steroid Hormone ◽  
Natural Infection ◽  
Placental Tissue ◽  
Selective Inhibition ◽  
Inhibitory Effect ◽  
Content Type ◽  
Direct Inhibitory Effect

ABSTRACT Coxiella burnetii is a zoonotic bacterial obligate intracellular parasite and the cause of query (Q) fever. During natural infection of female animals, C. burnetii shows tropism for the placenta and is associated with late-term abortion, at which time the pathogen titer in placental tissue can exceed one billion bacteria per gram. During later stages of pregnancy, placental trophoblasts serve as the major source of progesterone, a steroid hormone known to affect the replication of some pathogens. During infection of placenta-derived JEG-3 cells, C. burnetii showed sensitivity to progesterone but not the immediate precursor pregnenolone or estrogen, another major mammalian steroid hormone. Using host cell-free culture, progesterone was determined to have a direct inhibitory effect on C. burnetii replication. Synergy between the inhibitory effect of progesterone and the efflux pump inhibitors verapamil and 1-(1-naphthylmethyl)-piperazine is consistent with a role for efflux pumps in preventing progesterone-mediated inhibition of C. burnetii activity. The sensitivity of C. burnetii to progesterone, but not structurally related molecules, is consistent with the ability of progesterone to influence pathogen replication in progesterone-producing tissues.

scholarly journals Coxiella burnetii Intratracheal Aerosol Infection Model in Mice, Guinea Pigs, and Nonhuman Primates

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
A. E. Gregory ◽  
E. J. van Schaik ◽  
K. E. Russell-Lodrigue ◽  
A. P. Fratzke ◽  
J. E. Samuel
Keyword(s):  
Animal Models ◽  
Coxiella Burnetii ◽  
Guinea Pigs ◽  
Nonhuman Primates ◽  
Q Fever ◽  
Febrile Illness ◽  
Cell Vaccine ◽  
Infection Model ◽  
Atypical Pneumonia ◽  
Content Type

ABSTRACT Coxiella burnetii, the etiological agent of Q fever, is a Gram-negative bacterium transmitted to humans by inhalation of contaminated aerosols. Acute Q fever is often self-limiting, presenting as a febrile illness that can result in atypical pneumonia. In some cases, Q fever becomes chronic, leading to endocarditis that can be life threatening. The formalin-inactivated whole-cell vaccine (WCV) confers long-term protection but has significant side effects when administered to presensitized individuals. Designing new vaccines against C. burnetii remains a challenge and requires the use of clinically relevant modes of transmission in appropriate animal models. We have developed a safe and reproducible C. burnetii aerosol challenge in three different animal models to evaluate the effects of pulmonary acquired infection. Using a MicroSprayer aerosolizer, BL/6 mice and Hartley guinea pigs were infected intratracheally with C. burnetii Nine Mile phase I (NMI) and demonstrated susceptibility as determined by measuring bacterial growth in the lungs and subsequent dissemination to the spleen. Histological analysis of lung tissue showed significant pathology associated with disease, which was more severe in guinea pigs. Infection using large-particle aerosol (LPA) delivery was further confirmed in nonhuman primates, which developed fever and pneumonia. We also demonstrate that vaccinating mice and guinea pigs with WCV prior to LPA challenge is capable of eliciting protective immunity that significantly reduces splenomegaly and the bacterial burden in spleen and lung tissues. These data suggest that these models can have appreciable value in using the LPA delivery system to study pulmonary Q fever pathogenesis as well as designing vaccine countermeasures to C. burnetii aerosol transmission.

scholarly journals Detection of Coxiella burnetii DNA in the environment during and after a large Q fever epidemic in the Netherlands

2013 ◽  
Vol 114 (5) ◽  
pp. 1395-1404 ◽  
Author(s):  
A. de Bruin ◽  
I. Janse ◽  
M. Koning ◽  
L. de Heer ◽  
R.Q.J. van der Plaats ◽  
...  
Keyword(s):  
The Netherlands ◽  
Coxiella Burnetii ◽  
Q Fever

scholarly journals Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Megan E. Reller ◽  
J. Stephen Dumler
Keyword(s):  
Quantitative Pcr ◽  
Scrub Typhus ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Epidemiologic Studies ◽  
Qpcr Assay ◽  
Spotted Fever Group ◽  
Analytical Sensitivity ◽  
Content Type ◽  
Real Time Quantitative Pcr

ABSTRACT Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

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