Detection of Coxiella burnetii DNA on small-ruminant farms during a Q fever outbreak in the Netherlands

During 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 of Coxiella burnetii to humans is thought to occur primarily via aerosols, although available data on C. burnetii in 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 of C. burnetii DNA in vaginal swabs, manure, surface area swabs, milk unit filters, and aerosols. Four of these farms had a positive status during the Coxiella burnetii bulk 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 to C. burnetii and, to a lesser extent, on farms positive by bulk milk monitoring, generally higher proportions of positive samples and higher levels of C. burnetii DNA within positive samples were observed than on the control farms. The relatively high levels of C. burnetii DNA 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 of C. burnetii to humans.     

Detection of Coxiella burnetii from Dust in a Barn Housing Dairy Cattle

We attempted to detect Coxiella burnetii in dust samples collected from a barn housing dairy cattle by the polymerase chain reaction (PCR) method. Ten dust samples (five from ventilation fans and five from crossbeams) were collected from two areas in a barn on a farm near Sapporo, Hokkaido. C. burnetii was detected in 5 of the 10 dust samples. It was believed that aerial contamination by C. burnetii occurred in the barn.     

Genome Sequence of Coxiella burnetii 109, a Doxycycline-Resistant Clinical Isolate

Coxiella burnetii 109, with a 2.03-Mb genome, is a doxycycline-resistant human isolate that was isolated from the cardiac valve of a German male patient with Q fever endocarditis who died during the course of the treatment due to the bacterium''s resistance to doxycycline. This new genome can be useful for future comparative genomic or Q fever studies.     

Improved Method for Purification of Bacterial DNA from Bovine Milk for Detection of Brucella spp. by PCR

Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk.     

Detection of Coxiella burnetii DNA in inhalable airborne dust samples from goat farms after mandatory culling

Coxiella burnetii is thought to infect humans primarily via airborne transmission. However, air measurements of C. burnetii are sparse. We detected C. burnetii DNA in inhalable and PM10 (particulate matter with an aerodynamic size of 10 μm or less) dust samples collected at three affected goat farms, demonstrating that low levels of C. burnetii DNA are present in inhalable size fractions.     

Detection of Coxiella burnetii specific DNA in blood samples from Japanese patients with chronic nonspecific symptoms by nested polymerase chain reaction

Sprague Dawley rats, previously infected with Phase-I Bordetella pertussis , developed more severe abnormal respiratory sounds than normal animals, but not coughing, when exposed to aerosolized capsaicin, one of several cough-inducing agents tested. Stethoscope examination suggested that greater production of pulmonary mucus might be occurring after capsaicin challenge of the infected animals, compared to the uninfected controls. Rats of three other strains gave characteristically different responses from the Sprague Dawleys. The administration of capsaicin to B. pertussis -infected rats may provide useful insights into the pathophysiology of excess mucus secretion in human pertussis.     

Detection of Coxiella burnetii in complex matrices by using multiplex quantitative PCR during a major Q fever outbreak in The Netherlands

Q fever, caused by Coxiella 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 of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores 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 targets icd, com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA 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 the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.     

A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay

Aims: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small‐cell variant (SCV) by real‐time PCR (qPCR) in clinical samples. Methods and Results: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. Conclusions: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large‐cell and small‐cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. Significance and impact of study: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.     

Real-Time PCR for Measles Virus Detection on Clinical Specimens with Negative IgM Result in Morocco

Since the confirmation of measles cases represents an important indicator regarding the performance of the measles-elimination program, the aim of this study was to evaluate the effectiveness of the routine procedures followed in Morocco for the laboratory confirmation of measles cases. Suspected cases reported between January 2010 and December 2012 were assessed for the timeliness of the sample collection, occurrence of measles clinical symptoms, and the results of the laboratory diagnoses. For 88% of the 2,708 suspected cases, a clinical specimen was collected within 7d of rash onset, of which 50% were IgM-positive and 2.6% were equivocal. The measles symptoms were reported in 91.4% of the cases; the occurrence of symptoms showed a positive association with the serological results (odds ratio [OR] = 2.9883, 95% confidence interval [CI] 2.2238–4.0157). Of the negative samples, 52% (n = 116) tested positive by real-time polymerase chain reaction (PCR). These results are in favor of using molecular detection to complement serological diagnosis in the context of measles surveillance approach in Morocco. In addition, the introduction of additional laboratory methods for differential diagnosis is required for the final classification of suspected cases with maculopapular rash and fever in the context of the measles elimination program.     

Presence of Coxiella burnetii DNA in the Environment of the United States, 2006 to 2008

Coxiella burnetii is an obligate intracellular bacterium that causes the zoonotic disease Q fever. Because C. burnetii is highly infectious, can survive under a variety of environmental conditions, and has been weaponized in the past, it is classified as a select agent and is considered a potential bioweapon. The agent is known to be present in domestic livestock and in wild animal populations, but the background levels of C. burnetii in the environment have not been reported. To better understand the amount of C. burnetii present in the environment of the United States, more than 1,600 environmental samples were collected from six geographically diverse parts of the United States in the years 2006 to 2008. DNA was purified from these samples, and the presence of C. burnetii DNA was evaluated by quantitative PCR of the IS1111 repetitive element. Overall, 23.8% of the samples were positive for C. burnetii DNA. The prevalence in the different states ranged from 6 to 44%. C. burnetii DNA was detected in locations with livestock and also in locations with primarily human activity (post offices, stores, schools, etc.). This study demonstrates that C. burnetii is fairly common in the environment in the United States, and any analysis of C. burnetii after a suspected intentional release should be interpreted in light of these background levels. It also suggests that human exposure to C. burnetii may be more common than what is suggested by the number of reported cases of Q fever.The Gram-negative obligate intracellular bacterium Coxiella burnetii can infect humans and cause Q fever, an acute febrile illness (15, 17). Most cases of Q fever have fairly nonspecific symptoms, such as high fever, headache, myalgia, cough, and fatigue (29). Over one-third of patients may show signs of pneumonia or hepatitis (17). Acute cases typically resolve in 1 to 2 weeks, but a small percentage of Q fever cases result in a chronic infection that can present as endocarditis and be life-threatening (12).Q fever occurs worldwide, and numerous natural outbreaks have been reported in the United States (2, 23, 25) and other countries (5, 11, 18, 20, 22, 24). An ongoing natural outbreak in the Netherlands resulted in more than 2,000 cases of Q fever from 2007 to 2009 (27). In the United States Q fever became a nationally notifiable disease in 1999, and increasing numbers of cases have been reported to the CDC in recent years. However, the highest number of annual cases in the United States so far has been 171, reported in 2007 (8). Although this is a fairly small number of reported cases, it is possible that the number of actual cases in the United States is much higher. The relatively nonspecific nature of Q fever symptoms makes the disease difficult to diagnose, and people infected with C. burnetii are likely to show a diversity of symptoms with variable severity. The idea that Q fever is underreported is supported by our recent data using serum samples from the National Health and Nutrition Examination Survey (NHANES) to determine that the seroprevalence in the United States among people who are ≥20 years old is 3.1% (1).A common mechanism for people to become infected with C. burnetii is the inhalation of aerosolized bacteria. Potential sources for aerosolized C. burnetii are livestock and other animals. It is known that many herds of livestock are infected with C. burnetii and that seroprevalence rates in a variety of wild animal species can be quite high (17). Infected livestock herds do not typically show clinical signs of infection, but surges in abortion rates have been reported, particularly with goats (9, 10, 17). It is known that C. burnetii can replicate to high levels in the placenta of infected animals and that infectious C. burnetii can be spread to humans during parturition (9). The prevalence of C. burnetii in animals makes contact with animals a likely risk factor for Q fever. For example, the ongoing Q fever outbreak in the Netherlands has been linked to Q fever infections in goat farms (27), and we have recently found that 22.2% of a group of 508 veterinarians had antibodies against C. burnetii, a much higher seroprevalence than in the general U.S. population (31).C. burnetii exists as a replicating large-cell variant (LCV), but nonreplicating bacteria can form a more stable small-cell variant (SCV) (4). Although it is not an endospore, the SCV form of Coxiella is known to be very stable under a variety of conditions (16). C. burnetii is also highly infectious, with a dose of 1 to 10 organisms capable of causing Q fever in humans (30). These unique features of C. burnetii, along with its aerosol route of transmission, have led to the designation of C. burnetii as a category B bioterrorism weapon and inclusion on the list of select agents. The potential for the use of C. burnetii as a bioweapon was explored in detail by the U.S. bioweapons program of the 1950s and 1960s (26). Although not typically lethal, C. burnetii is considered a threat due to its ability to cause widespread debilitating illness. Indeed, many U.S. soldiers returning from Iraq between 2005 and 2008 suffered from Q fever while deployed (6, 7). These cases are suspected to be naturally acquired infections.The potential for both intentional releases and natural outbreaks makes it important to understand the presence of C. burnetii in the environment. Investigations of the source of Q fever cases will include a determination of the presence of C. burnetii in the environment from which the bacteria may have been acquired. The purpose of this study was to analyze a large number of samples across a wide geographic distribution in the United States and to establish a baseline for the presence of C. burnetii in different regions of the country.     

用逆转录套式PCR检测临床标本中的腮腺炎病毒RNA

流行性腮腺炎是儿童常见呼吸道传染病,并发症多为睾丸炎、无菌脑炎等。个别患者甚至因严重的脑炎并发症而死亡［１］。故有必要对患者进行早期病原诊断,以利于早期治疗。腮腺炎病毒ＲＮＡ中小疏水蛋白（ＳＨ）基因作为高变区在不同毒株的分子特性鉴别中比其它基因片段更...     

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

Background  

Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals.     

Detection of Coxiella burnetii in cow's milk by PCR-enzyme-linked immunosorbent assay combined with a novel sample preparation method.

The use of an adequate concentration of Triton X-100 enhanced immunomagnetic separation of Coxiella burnetii from milk. PCR-enzyme-linked immunosorbent assay (PCR-ELISA) could detect coxiellas more sensitively than could conventional PCR. PCR-ELISA is therefore thought to be suitable for the simultaneous assay of a large number of samples. However, the number of cows from which raw milk tested positive for coxiellas by PCR-ELISA was inconsistent with that found with the antibody to coxiella by indirect immunofluorescence assay. The inconsistency is thought to be associated with the differences in the infectious route, infectious dose, or the timing of yielding the antibody and the period of duration of the antibody.     

The Sero-epidemiology of Coxiella burnetii in Humans and Cattle,Western Kenya: Evidence from a Cross-Sectional Study

Evidence suggests that the intracellular bacterial pathogen Coxiella burnetii (which causes Q fever) is widespread, with a near global distribution. While there has been increasing attention to Q fever epidemiology in high-income settings, a recent systematic review highlighted significant gaps in our understanding of the prevalence, spatial distribution and risk factors for Q fever infection across Africa. This research aimed to provide a One Health assessment of Q fever epidemiology in parts of Western and Nyanza Provinces, Western Kenya, in cattle and humans. A cross-sectional survey was conducted: serum samples from 2049 humans and 955 cattle in 416 homesteads were analysed for C. burnetii antibodies. Questionnaires covering demographic, socio-economic and husbandry information were also administered. These data were linked to environmental datasets based on geographical locations (e.g., land cover). Correlation and spatial-cross correlation analyses were applied to assess the potential link between cattle and human seroprevalence. Multilevel regression analysis was used to assess the relationships between a range of socio-economic, demographic and environmental factors and sero-positivity in both humans and animals. The overall sero-prevalence of C. burnetii was 2.5% in humans and 10.5% in cattle, but we found no evidence of correlation between cattle and human seroprevalence either within households, or when incorporating spatial proximity to other households in the survey. Multilevel modelling indicated the importance of several factors for exposure to the organism. Cattle obtained from market (as opposed to those bred in their homestead) and those residing in areas with lower precipitation levels had the highest sero-prevalence. For humans, the youngest age group had the highest odds of seropositivity, variations were observed between ethnic groups, and frequent livestock contact (specifically grazing and dealing with abortion material) was also a risk factor. These results illustrate endemicity of C. burnetii in western Kenya, although prevalence is relatively low. The analysis indicates that while environmental factors may play a role in cattle exposure patterns, human exposure patterns are likely to be driven more strongly by livestock contacts. The implication of livestock markets in cattle exposure risks suggests these may be a suitable target for interventions.     

Isolation of a Coxiella burnetii strain that has low virulence for mice from a patient with acute Q fever

Coxiella burnetii was isolated from a patient with Q fever. It could not be determined whether this was an imported case or an indigenous one. Identification of the isolate was made by electron microscopic morphology and the indirect fluorescent antibody test with convalescent-phase serum from a Q fever patient having a known titer of antibody to C. burnetii. The isolated strain, named TK-1, caused no symptoms in ddY and BALB/c mice except when the mice were treated with cyclophosphamide.     

Preparation of single-stranded DNA from PCR products with streptavidin magnetic beads

The preparation of single-stranded DNA from double-stranded PCR products is an essential step in the identification of aptamers by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The most widely used method for producing single-stranded DNA is alkaline denaturation of biotinylated PCR products attached to streptavidin-coated magnetic beads. Recently, it has been suggested that this method may be unsuitable due to the release of interfering amounts of streptavidin and biotinylated DNA. In this article, the alkaline method is compared with a thermal method that is known to release significant amounts of streptavidin and biotinylated DNA. Results show that trace amounts of streptavidin and biotinylated DNA are released in the alkaline method, but this can be curtailed by preconditioning the beads in aqueous sodium hydroxide. The main product in the alkaline method is single-stranded DNA, which is produced in high yield.