A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.     

Animal models in Q fever: pathological responses of inbred mice to phase I Coxiella burnetii

The susceptibility of inbred strains of mice to infection by phase I Coxiella burnetii, the aetiological agent of Q fever, was investigated by evaluating morbidity, mortality, antibody production and in vitro proliferative responses of splenic lymphocytes. Among the 47 strains of mice tested for morbidity and mortality to C. burnetii infection, 33 were resistant, 10 were of intermediate sensitivity, and four were sensitive. A/J mice exhibited the highest mortality, and surviving mice of this strain yielded high concentrations of viable rickettsiae from essentially all organs for more than 3 weeks after inoculation. However, A/J mice developed a protective immune response after vaccination with inactivated C. burnetii cells. Induction of gross pathological responses and antibody production were similar in sensitive mice (strain A/J) and resistant mice (strain C57BL/6J). The LD50 of phase I C. burnetii for A/J mice was about 1000-fold lower than that for the more resistant C57BL/6J mice. Mice of both strains developed antibody titres against phase I cells, phase II cells, and phase I lipopolysaccharide after the injection of one or more viable phase I organisms of C. burnetii; five or more rickettsiae caused splenomegaly that was almost proportional to the infecting dose. Suppression of in vitro proliferative responses of splenic lymphocytes to concanavalin A, a T-cell mitogen, was apparent after infection of sensitive A/J mice with as few as one to five phase I micro-organisms. However, suppression of proliferation of splenic lymphocytes from resistant C57BL/6J mice required 10(7) phase I C. burnetii.     

Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.     

Profiling the humoral immune response of acute and chronic Q fever by protein microarray

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.     

Effects of Coxiella burnetii on MAPKinases phosphorylation

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.     

Effects of X-irradiation on the immune response of guinea pigs to Q fever vaccine

The immune response of guinea pigs to Q fever vaccine following 75 to 250 R (60 to 180 rads) of acute whole-body irradiations was investigated. Complement-fixing (CF) antibody titers and protection against febrile response to challenge with virulent Coxiella burnetii were studied. Exposures ranging from 75 to 250 R, 24 hours prior to inoculation, did not detectably alter the CF antibody response. Similar results were observed with 175 R delivered 48 or 72 hours before immunization. Protection against febrile response to challenge with 10(3) median fever doses of C. burnetii was seen in animals irradiated with 175 R, 24 or 72 hours before immunization. Significant protection was detectable at 14, 21, and 42 days after immunization in both irradiated and nonirradiated animals. Acute irradiation of the degree studied increases the mortality in normal animals infected 15 to 17 days later with virulent C. burnetii. The lethal effect could be prevented by use of Q fever vaccine.     

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.     

Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.     

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

Developing a new clinical tool for diagnosing chronic Q fever: the Coxiella ELISPOT

Definitively establishing a clinical diagnosis of chronic Q fever remains challenging, as the diagnostic performance of both conventional serological tests and PCR is limited. Given the importance of an early diagnosis of chronic Q fever, there is a need for a reliable diagnostic test. We developed an enzyme-linked immunospot assay to measure Coxiella burnetii (C.?burnetii)-specific T-cell responses (Coxiella ELISPOT) to both phase I and phase II antigens and tested convalescent Q fever patients (without chronic disease, n?=?9) and patients with an established diagnosis of chronic Q fever (n?=?3). The Coxiella ELISPOT adequately identified convalescent Q fever patients from healthy controls by demonstrating C.?burnetii-specific T-cell interferon-γ production to both phase I and phase II antigens. Compared to convalescent Q fever patients, chronic Q fever patients showed a distinct Coxiella ELISPOT profile characterized by a much higher spot count for both phase I and phase II (18-fold for phase II, 8-fold higher for phase I) and a consistent shift towards more phase I reactivity. The diagnostic potential of the Coxiella ELISPOT is promising and warrants further investigation.     

Serological evidence that the Q fever agent (Coxiella burnetii) has spread widely among dairy cattle of Japan.

Serological examination of bovine and human sera for antibodies against Coxiella burnetti was carried out by the immunofluorescence technique. Twenty to 30% of the cows examined were antibody-positive. Sera from two veterinarians also had antibody against C. burnetii. These results suggest an increase in the number of infected cows with C. burnetii in Japan since 1954, and also imply the possibility of the prevalence of acute Q fever in the human population, which had been underestimated and undiagnosed for the last three decades.     

Detecting and measuring small numbers of viable Coxiella burnetii

Coxiella burnetii is an acidophilic, intracellular bacterium that causes the human disease Q fever. In some studies, it is important to distinguish between viable and nonviable C.?burnetii. We compared four methods for detecting and measuring viable C.?burnetii in biological samples as follows: growth in two different cell culture lines, infection of severe combined immunodeficient (SCID) mice (leading to death) and infection of SCID mice with detection of C.?burnetii in their spleen (after euthanasia at day 50 postinfection). Two isolates of C.?burnetii were used ('Henzerling' and 'Arandale'). Our in-house qPCR assay for C.?burnetii DNA was used as a control. SCID mouse inoculation was more sensitive than cell culture. The assay that detected C.?burnetii in SCID mouse spleens was slightly more sensitive than SCID mice deaths alone. Approximately one viable C.?burnetii cell could be detected by this method, making it suitable for determining the viability of C.?burnetii in a sample.     

Identification of CD4+ T cell epitopes in C. burnetii antigens targeted by antibody responses

Coxiella burnetii is an obligate intracellular gram-negative bacterium that causes acute Q fever and chronic infections in humans. A killed, whole cell vaccine is efficacious, but vaccination can result in severe local or systemic adverse reactions. Although T cell responses are considered pivotal for vaccine derived protective immunity, the epitope targets of CD4(+) T cell responses in C. burnetii vaccination have not been elucidated. Since mapping CD4(+) epitopes in a genome with over 2,000 ORFs is resource intensive, we focused on 7 antigens that were known to be targeted by antibody responses. 117 candidate peptides were selected from these antigens based on bioinformatics predictions of binding to the murine MHC class II molecule H-2 IA(b). We screened these peptides for recognition by IFN-γ producing CD4(+) T cell in phase I C. burnetii whole cell vaccine (PI-WCV) vaccinated C57BL/6 mice and identified 8 distinct epitopes from four different proteins. The identified epitope targets account for 8% of the total vaccination induced IFN-γ producing CD4(+) T cells. Given that less than 0.4% of the antigens contained in C. burnetii were screened, this suggests that prioritizing antigens targeted by antibody responses is an efficient strategy to identify at least a subset of CD4(+) targets in large pathogens. Finally, we examined the nature of linkage between CD4(+) T cell and antibody responses in PI-WCV vaccinated mice. We found a surprisingly non-uniform pattern in the help provided by epitope specific CD4(+) T cells for antibody production, which can be specific for the epitope source antigen as well as non-specific. This suggests that a complete map of CD4(+) response targets in PI-WCV vaccinated mice will likely include antigens against which no antibody responses are made.     

Coxiella burnetii stimulates production of RANTES and MCP-1 by mononuclear cells: modulation by adhesion to endothelial cells and its implication in Q fever

Q fever is an infectious disease caused by Coxiella burnetii, which may become chronic when cytokine network and cell-mediated immune responses are altered. Chemokines, such as Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES, CCL5) and Monocyte Chemoattractant Protein-1 (MCP-1, CCL2), are specialized in the trafficing of peripheral blood mononuclear cells (PBMC), and are associated with T cell polarization that is essential for intracellular survival of C. burnetii. The present study investigated whether or not the infection status (no infection and acute or chronic infection with C. burnetii) of donors, affected the production of the two chemokines by PBMC with or without stimulation with virulent and avirulent C. burnetii. Our findings indicate that in vitro exposure to virulent or avirulent C. burnetii stimulated the production of RANTES and MCP-1 in PBMC obtained from healthy adults. The co-cultivation of endothelial cells and human PBMC resulted in an increased production of MCP-1 and the up-regulation of RANTES, which were contact-dependent. Unstimulated PBMC from patients with acute or chronic Q fever overproduced MCP-1. Interestingly, the addition of C. burnetii resulted in an increased production of RANTES and MCP-1 by PBMC obtained from patients with chronic Q fever, and the co-cultivation of PBMC with endothelial cells amplified increased production of chemokines. Circulating levels of RANTES and MCP-1 were also increased in chronic Q fever. We suggest that the overproduction of RANTES and MCP-1 secondary to the contact of PBMC with endothelium may perpetuate exaggerated inflammatory responses leading to inappropriate PBMC trafficking and to the pathogenesis of Q fever.     

Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype.     

Serotyping Coxiella burnetii isolates from acute and chronic Q fever patients by using monoclonal antibodies

Abstract Four mouse monoclonal antibodies reacting with Coxiella burnetii lipopolysaccharide antigens were produced and used in serotyping 17 C. burnetii isolates from acute Q fever and Q fever endocarditis patients in France. Two monoclonal antibodies (1B2 and 3B6) were considered specific for the Priscilla strain, a representative of Q fever endocarditis isolates, and did not react with the Nine Mile strain, which is representative of acute Q fever isolates. Monoclonal antibodies Nos. 1B2 and 3B6 reacted with 75% (3/4) acute Q fever isolates and 85% (11/13) of endocarditis isolates from France. It is reasonable to conclude that Priscilla-like strains cause both acute Q fever and Q fever endocarditis. The hypothesis that Priscilla-like strains only are associated with Q fever endocarditis should be reconsidered.     

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.     

Plaque Assay for Q Fever and Scrub Typhus Rickettsiae

The plaque assay procedure developed for spotted fever and typhus group rickettsiae is also appropriate for scrub typhus and Q fever rickettsiae. The plaque titers of suspensions of Rickettsia tsutsugamushi and Coxiella burnetii compared favorably with end points obtained by titrations in mice.     

First case of Q fever endocarditis in Croatia and a short review

We present a 70-year-old man from Dalmatia, Croatia, with a history of prolonged high fever diagnosed as Q fever endocarditis. As far as we know, this is the first case of chronic Q fever in Croatia. The treatment was started as for culture-negative endocarditis, but was without clinical response. After significantly high anti-phase I IgG plus IgA antibodies titers to Coxiella burnetii were shown, the initial treatment with doxycycline was changed and ciprofloxacin was started with good clinical response.