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Local and systemic reactogenic responses to Q-VAX have prevented licensing of this vaccine outside of Australia. These reactogenic responses occur in previously sensitized individuals and have not been well defined at the cellular level, in part because many studies have been done in guinea pigs that have limited molecular tools. We previously characterized a mouse model of reactogenicity where local reaction sites showed an influx of CD8+ and IFNγ-expressing IL17a+ CD4+ T cells consistent with a Th1 delayed-type hypersensitivity. In this study, we determined, using depletion and adoptive transfer experiments, that both anti- Coxiella antibodies and CD4+ T cells were essential for localized reactions at the site of vaccination. Furthermore, IFNγ depletion showed significant histological changes at the local reaction sites demonstrating the essential nature of this cytokine to reactogenicity. In addition to the cells and cytokines required for this response, we determined that whole cell vaccine (WCV) material remained at the site of vaccination for at least 26 weeks post-injection. Transmission electron microscopy (TEM) of these sites demonstrated intact rod-shaped bacteria at 2 weeks post-injection and partially degraded bacteria within macrophages at 26 weeks post-injection. Finally, because small cell variants (SCVs) are an environmentally stable form, we determined that local reactions were more severe when the WCV material was prepared with higher levels of SCVs compared to typical WCV or with higher levels of large cell variant (LCV). These studies support the hypothesis that antigen persistence at the site of injection contributes to this reactogenicity and that anti- Coxiella antibodies, CD4+ T cells, and IFNγ each contribute to this process., Competing Interests: The authors declare no conflict of interest.

Coxiella burnetii is an obligate intracellular Gram-negative bacterium that causes acute and chronic Q fever in humans. Acute Q fever is usually a flu-like, self-limiting or treatable illness, but some infections can turn into a severe and sometimes fatal chronic disease. There is currently no FDA-approved vaccine available for the prevention of human Q fever in the US, development of a safe and effective vaccine for the prevention of human Q fever remains an important goal for public health. However, there is a fundamental gap in knowledge regarding the mechanism of protective immunity against C. burnetii infection. To understand the mechanism of C. burnetii infection induced protective immunity, we examined if C. burnetii Nine Mile phase I (NMI) infection induces protection against C. burnetii reinfection in mice. Our results indicate that NMI-infected mice conferred significant protection against C. burnetii reinfection. We also found that NMI infection derived protection did not depend on the routes of infection and antibodies are required for NMI infection derived protection. In addition, NMI infection elicited a comparable level of protection in Wild type, CD4 + T cell deficient, and CD8 + T cell deficient mice, partial protection in B cell deficient mice but no protection in T cell deficient mice. These results suggest that both B cells and T cells are required for NMI-infection derived protection, but T cells may play a critical role. Therefore, the new generation vaccine for the prevention of human Q fever should be focused on boosting both humoral and T cell immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alam, Kumaresan, Palanisamy, Zhang, Seshu, Xiong and Zhang.)

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii , which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1 -/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1 +/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1 -/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1 +/- macrophages. Uptake of added isomers into Acod1 -/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Siddique, Kellermeier, Ölke, Zhao, Büssow, Oefner, Lührmann, Dettmer and Lang.)

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii , but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii -infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8 -/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii , coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii , this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8 -/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors., Competing Interests: The authors declare no conflict of interest.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic bacteria of global public health significance. The organism has a complex, diverse, and relatively poorly understood animal reservoir but there is increasing evidence that macropods play some part in the epidemiology of Q fever in Australia. The aim of this cross-sectional survey was to estimate the animal- and tissue-level prevalence of coxiellosis amongst eastern grey (Macropus giganteus) and red (Osphranter rufus) kangaroos co-grazing with domestic cattle in a Q fever endemic area in Queensland. Serum, faeces and tissue samples from a range of organs were collected from 50 kangaroos. A total of 537 tissue samples were tested by real-time PCR, of which 99 specimens from 42 kangaroos (84% of animals, 95% confidence interval [CI], 71% to 93%) were positive for the C. burnetii IS1111 gene when tested in duplicate. Twenty of these specimens from 16 kangaroos (32%, 95% CI 20% to 47%) were also positive for the com1 or htpAB genes. Serum antibodies were present in 24 (57%, 95% CI 41% to 72%) of the PCR positive animals. There was no statistically significant difference in PCR positivity between organs and no single sample type consistently identified C. burnetii positive kangaroos. The results from this study identify a high apparent prevalence of C. burnetii amongst macropods in the study area, albeit seemingly with an inconsistent distribution within tissues and in relatively small quantities, often verging on the limits of detection. We recommend Q fever surveillance in macropods should involve a combination of serosurveys and molecular testing to increase chances of detection in a population, noting that a range of tissues would likely need to be sampled to confirm the diagnosis in a suspect positive animal., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tolpinrud et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Vγ9Vδ2 T cells have been reported to participate to the immune response against infectious diseases such as the Q fever caused by Coxiella burnetii infection. Indeed, the number and proportion of Vγ9Vδ2 T cells are increased during the acute phase of Q fever. Human Vγ9Vδ2 T cell responses are triggered by phosphoantigens (pAgs) produced by pathogens and malignant cells, that are sensed via the membrane receptors butyrophilin-3A1 (BTN3A1) and -2A1 (BTN2A1). Here, by using CRISPR-Cas9 inactivation in THP-1 cells, we show that BTN3A and BTN2A are required to Vγ9Vδ2 T cell response to C. burnetii infection, though not directly involved in the infection process. Furthermore, C. burnetii -infected monocytes display increased BTN3A and BTN2A expression and induce Vγ9Vδ2 T cell activation that can be inhibited by specific antagonist mAb. More importantly, we show that the antimicrobial functions of Vγ9Vδ2 T cells towards C. burnetii are enhanced in the presence of an BTN3A activating antibody. This supports the role of Vγ9Vδ2 T cells in the control of C. burnetii infection and argues in favor of targeting these cells as an alternative treatment strategy for infectious diseases caused by intracellular bacteria., Competing Interests: DO is cofounder and shareholder of Imcheck Therapeutics, Emergence Therapeutics, Alderaan Biotechnology and Stealth IO. CC, EF, MG, MF, and LM are employees and shareholders of Imcheck Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The authors declare that this study received funding from ImCheck Therapeutics. The funder had the following involvement in the study: study design, collection, analysis, interpretation of data and the writing of this article., (Copyright © 2022 Gay, Mezouar, Cano, Foucher, Gabriac, Fullana, Madakamutil, Mège and Olive.)

Background: Chronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue., Methods: Multiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune systems., Results: Q fever AAAs and atherosclerotic AAAs contained similar numbers of CD68 + macrophages and CD3 + T cells. However, in Q fever AAAs, the number of CD68 + CD206 + M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8 + cytotoxic T cells and CD3 + CD8 - FoxP3 + regulatory T cells. Finally, Q fever AAAs did not contain any well-defined granulomas., Conclusions: These findings demonstrate that despite the presence of pro-inflammatory effector cells, persistent local infection with C. burnetii is associated with an immune-suppressed microenvironment., Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716]., Competing Interests: KC, AS, TS, MG, LV, AJ, PP, RV, PW, CB, MS, KH, Mv, JD, RV No competing interests declared, JV Senior editor, eLife, (© 2022, Cortenbach et al.)

Coxiella burnetii is a bacterial pathogen that replicates within host cells by establishing a membrane-bound niche called the Coxiella -containing vacuole. Biogenesis of this compartment requires effectors of its Dot/Icm type IV secretion system. A large cohort of such effectors has been identified, but the function of most of them remain elusive. Here, by a cell-based functional screening, we identified the effector Cbu0513 (designated as CinF) as an inhibitor of NF-κB signaling. CinF is highly similar to a fructose-1,6-bisphosphate (FBP) aldolase/phosphatase present in diverse bacteria. Further study reveals that unlike its ortholog from Sulfolobus tokodaii , CinF does not exhibit FBP phosphatase activity. Instead, it functions as a protein phosphatase that specifically dephosphorylates and stabilizes IκBα. The IκBα phosphatase activity is essential for the role of CinF in C. burnetii virulence. Our results establish that C. burnetii utilizes a protein adapted from sugar metabolism to subvert host immunity., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Q fever is caused by the intracellular bacterium Coxiella burnetii , for which there is no approved vaccine in the United States. A formalin-inactivated whole-cell vaccine (WCV) from virulent C. burnetii NMI provides single-dose long-lived protection, but concerns remain over vaccine reactogenicity. We therefore sought an alternate approach by purifying native C. burnetii antigens from the clonally derived avirulent NMII strain. A soluble bacterial extract, termed Sol II, elicits high-titer, high-avidity antibodies and induces a CD4 T cell response that confers protection in naive mice. In addition, Sol II protects against pulmonary C. burnetii challenge in three animal models without inducing hypersensitivity. An NMI-derived extract, Sol I, enhances protection further and outperforms the WCV gold standard. Collectively, these data represent a promising approach to design highly effective, non-reactogenic Q fever vaccines., Competing Interests: J.E.S., E.J.V.S., and A.E.G. are named inventors of patent US 2019/0083598 A1., (© 2021 The Authors.)

Coxiella burnetii is an obligate intracellular bacterium that causes the human disease Q fever, which can manifest as an acute flu-like illness or a long-term chronic illness, such as endocarditis. Three genotypes (ST8, ST16, and ST20) of Coxiella burnetii are commonly found in the contemporary US and are associated with specific animal hosts. Although all three genotypes have been isolated from humans with Q fever, studies comparing virulence between C. burnetii sequence types have been rare. Here, groups of mice were infected via aerosol inoculation with isolates derived from cow's milk, environmental, animal, and human samples. Mice were monitored for weight loss and blood samples were takenweekly. Animals were euthanized at 2- and 12-weeks post-infection, and bacterial burden was determined for tissues by real-time PCR. The levels of anti-Coxiella antibodies and selected inflammatory cytokines were determined for serum samples. Weight loss and splenomegaly were observed in mice infected with ST20 and ST16 isolates but were absent in the mice infected with ST8 isolates. Bacterial concentrations in the tissues were lower in the ST8 isolates at 2 weeks post-infection relative to all other isolates. ST16 and ST20 isolates induced robust antibody and cytokine responses, while ST8 isolates produced significantly lower anti- C. burnetii titers early in the infection but saw increased titers in some animals several weeks post-infection. The data suggest that the ST8 isolates are less virulent in this mouse model, as they produce less robust antibody responses that are slow to develop, relative to the ST16 and ST20 isolates.

This study aimed to explore if viable C. burnetii avirulent Nine Mile phase II (NMII) can elicit protective immunity against virulent NM phase I (NMI) infection. Interestingly, mice immunized with viable NMII elicited significant protection against NMI infection at different time points post-immunization. Viable NMII induced a dose-dependent NMI-specific IgG response in mice, but all doses of NMII-immunized mice conferred a similar level of protection. Comparing different routes of immunization indicated that intranasally immunized mice showed significantly higher levels of protection than other immunization routes. The observation that viable NMII induced a similar level of long-term protection against NMI challenge as the formalin-inactivated NMI vaccine (PIV) suggests that viable NMII bacteria can induce a similar level of long-term protection against virulent NMI challenge as the PIV. Viable NMII also induced significant protection against challenge with virulent Priscilla and Scurry strains, suggesting that viable NMII can elicit broad protection. Immune sera and splenocytes from viable NMII-immunized mice are protective against NMI infection, but immune serum-receiving mice did not control NMI replication. Additionally, viable NMII conferred a comparable level of protection in wild-type, CD4 + T cell-deficient, and CD8 + T cell-deficient mice, and partial protection in B cell-deficient mice. However, NMII-immunized T cell-deficient mice were unable to prevent C. burnetii replication. Thus, both B cells and T cells are required for viable NMII-induced protective immunity but T cells may play a critical role. Collectively, this study demonstrates the feasibility of using avirulent NMII as a live attenuated vaccine against human Q fever., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kumaresan, Alam, Zhang and Zhang.)

For the zoonotic disease Q fever, serological analysis plays a dominant role in the diagnosis of Coxiella burnetii infection and in pre-screening for past exposure prior to vaccination. A number of studies suggest that assessment of C. burnetii -specific T-cell IFNγ responses may be a more sensitive tool to assess past exposure. In this study, we assessed the performance of a whole blood C. burnetii IFNγ release assay in comparison to serological detection in an area of high Q fever incidence in 2014, up to seven years after initial exposure during the Dutch Q fever outbreak 2007-2010. In a cohort of >1500 individuals from the Dutch outbreak village of Herpen, approximately 60% had mounted IFNγ responses to C. burnetii . This proportion was independent of the Coxiella strain used for stimulation and much higher than the proportion of individuals scored sero-positive using the serological gold standard immunofluorescence assay. Moreover, C. burnetii -specific IFNγ responses were found to be more durable than antibody responses in two sub-groups of individuals known to have sero-converted as of 2007 or previously reported to the municipality as notified Q fever cases. A novel ready-to-use version of the IFNγ release assay assessed in a subgroup of pre-exposed individuals in 2021 (10-14 years post exposure) proved again to be more sensitive than serology in detecting past exposure. These data demonstrate that C. burnetii -induced IFNγ release is indeed a more sensitive and durable marker of exposure to C. burnetii than are serological responses. In combination with a simplified assay version suitable for implementation in routine diagnostic settings, this makes the assessment of IFNγ responses a valuable tool for exposure screening to obtain epidemiological data, and to identify previously exposed individuals in pre-vaccination screens., Competing Interests: AG is a senior officer and shareholder of Innatoss Laboratories B.V., MK is currently employed and AS and MV were employed by Innatoss Laboratories B.V., which provides diagnostic screening for infectious diseases and has developed and owns the patent for the Q-detect™ IGRA for cellular immunity testing for Q fever. HPD was employed by Numerus GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Scholzen, de Vries, Duerr, Roest, Sluder, Poznansky, Kouwijzer and Garritsen.)

Coxiella burnetii , a highly adapted obligate intracellular bacterial pathogen and the cause of the zoonosis Q fever, is a reemerging public health threat. C. burnetii employs a Type IV secretion system (T4SS) to establish and maintain its intracellular niche and modulate host immune responses including the inhibition of apoptosis. Interactions between C. burnetii and caspase-1-mediated inflammasomes are not fully elucidated. This study confirms that C. burnetii does not activate caspase-1 during infection of mouse macrophages in vitro. C. burnetii -infected cells did not develop NLRP3 and ASC foci indicating its ability to avoid cytosolic detection. C. burnetii is unable to inhibit the pyroptosis and IL-1β secretion that is induced by potent inflammasome stimuli but rather enhances these caspase-1-mediated effects. We found that C. burnetii upregulates pro-IL-1β and robustly primes NLRP3 inflammasomes via TLR2 and MyD88 signaling. As for wildtype C. burnetii , T4SS-deficient mutants primed and potentiated NLRP3 inflammasomes. An in vivo model of pulmonary infection in C57BL/6 mice was developed. Mice deficient in NLRP3 or caspase-1 were like wildtype mice in the development and resolution of splenomegaly due to red pulp hyperplasia, and histologic lesions and macrophage kinetics, but had slightly higher pulmonary bacterial burdens at the greatest measured time point. Together these findings indicate that C. burnetii primes but avoids cytosolic detection by NLRP3 inflammasomes, which are not required for the clinical resistance of C57BL/6 mice. Determining mechanisms employed by C. burnetii to avoid cytosolic detection via NLRP3 inflammasomes will be beneficial to the development of preventative and interventional therapies for Q fever.

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii , a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX ® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii . While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses., Competing Interests: PF, DD, JF, LL, SJ, RN, and AJ own shares in Nanommune Inc. Nanommune does not sell the arrays described in this paper, nor funded any part of the work described herein. Neither Nanommune or its shareholders are likely to benefit from the results described in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fratzke, Jan, Felgner, Liang, Nakajima, Jasinskas, Manna, Nihesh, Maiti, Albin, Esser-Kahn, Davies, Samuel, Felgner and Gregory.)

Serology is essential for Q fever diagnostics, a disease caused by the bacterial pathogen Coxiella burnetii. The gold standard test is an immunofluorescence assay utilizing whole cell antigens, which are both dangerous and laborious to produce. Complexities of the antigen coupled with the subjective nature of the assay lead to decreased uniformity of test results and underscore the need for improved methodologies. Thirty-three C. burnetii proteins, previously identified as immunoreactive, were screened for reactivity to naturally infected goat serum. Based on reactivity, 10 proteins were analyzed in a secondary screen against human serum from healthy donors. Assay sensitivity and specificity ranged from 21 to 71% and 90 to 100%, respectively. Three promising antigens were identified based on receiver operating characteristic curve analysis (CBU_1718, CBU_0307, and CBU_1398). Five multiplex assays failed to outperform the individual proteins, with sensitivities and specificities ranging from 29 to 57% and 90 to 100%, respectively. Truncating the top antigen, CBU_1718, had no effect on specificity (90%); yet sensitivity decreased dramatically (71% to 21%). Through this study, we have expanded the subset of C. burnetii immunoreactive proteins validated by enzyme-linked immunosorbent assay and demonstrate the effect of novel antigen combinations and protein truncations on assay performance.

Natural killer (NK) cells are critically involved in the early immune response against various intracellular pathogens, including Coxiella burnetii and Chlamydia psittaci Chlamydia -infected NK cells functionally mature, induce cellular immunity, and protect themselves by killing the bacteria in secreted granules. Here, we report that infected NK cells do not allow intracellular multiday growth of Coxiella , as is usually observed in other host cell types. C. burnetii -infected NK cells display maturation and gamma interferon (IFN-γ) secretion, as well as the release of Coxiella -containing lytic granules. Thus, NK cells possess a potent program to restrain and expel different types of invading bacteria via degranulation. Strikingly, though, in contrast to Chlamydia , expulsed Coxiella organisms largely retain their infectivity and, hence, escape the cell-autonomous self-defense mechanism in NK cells., (Copyright © 2020 American Society for Microbiology.)

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA ::Tn or a Dot/Icm-defective dotA ::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen., Competing Interests: The authors declare no competing interest.

Several commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of phase II IgG or IgM antibodies against Coxiella burnetii were compared. In addition, an indirect immunofluorescence test was used as a confirmation test. In all, 70 serum samples for IgG and 43 serum samples for IgM were tested. The ELISAs showed large differences in sensitivity and specificity, which led to a partially high ratio of false-negative determinations. The most convincing test was PanBio from Abbott, which unfortunately can only test IgG but not IgM., (Copyright © 2020 American Society for Microbiology.)

Q fever is a worldwide zoonosis caused by Coxiella burnetii. Human Q fever is typically acquired through inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In this study, BALB/c mice were infected with C. burnetii via an intratracheal (IT) route using a non-invasive aerosol pulmonary delivery device to directly place the living C. burnetii organisms into the lungs of the mice. The bacterial loads, pathological lesions, and antibody and cellular responses were analyzed and compared with those of mice infected via an intraperitoneal (IP) route. Compared with mice infected via an IP route, mice infected via an IT route exhibited a higher bacterial load and more severe pathological lesions in the heart and lungs at days 3 and 7 post-infection (pi). The levels of interferon-γ and IL-12p70 in the serum of mice infected via the IT route were significantly higher than those of mice infected via the IP route at day 3 pi. In conclusion, this murine model of acute C. burnetii infection via IT inoculation closely resembles the natural route of C. burnetii infection than that of IP injection. Thus, this newly developed model will be useful for investigating the pathogenesis and immunity of C. burnetii aerosol infection, as well as for the evaluation of therapeutic drugs and preventive vaccines of Q fever., Competing Interests: The authors have declared that no competing interests exist.

Coxiella burnetii is an intracellular, gram-negative bacterium that causes the zoonosis Q fever. This disease typically presents as an acute flu-like illness with persistent, focalized infections occurring less frequently. Clinical outcomes of Q fever have been associated with distinct genomic groups of C. burnetii, suggesting that gene content is responsible for virulence potential. To investigate this hypothesis, the virulence of thirteen C. burnetii strains (representing genomic groups I-VI) was evaluated in a guinea pig infection model by intraperitoneal injection. Seven strains caused a sustained fever (at least two days ≥39.5°C) in at least half of the animals within each experimental group. At fourteen days post infection, animals were euthanized and additional endpoints were evaluated, including splenomegaly and serology. The magnitude of these endpoints roughly correlated with the onset, duration, and severity of fever. The most severe disease was caused by group I strains. Intermediate and no virulence were evidenced following infection with group II-V and group VI strains, respectively. Flow cytometric analysis of the mesenteric lymph nodes revealed decreased CD4 + T cell frequency following infection with highly virulent group I strains. These findings buttress the hypothesis that the pathogenic potential of C. burnetii strains correlates with genomic grouping. These data, combined with comparative genomics and genetic manipulation, will improve our understanding of C. burnetii virulence determinants.

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., (Copyright © 2019 American Society for Microbiology.)

Due to the atypical serological profile of some patients with primary Q fever infection who do not develop IgM against Coxiella burnetii , we developed an avidity test to distinguish recent or past infections. We tested 39 serum samples by immunofluorescence with conventional assay and after urea treatment from 26 patients at different stages of the disease. We observed a strong avidity in the 15 serum samples from patients with infections of >6 months and a low avidity for sera from patients with recent infections. A complete denaturation of the antibody-antigen complex was observed for patients for whom the time since the beginning of infection was <1 month and a mean of 2.06 ± 0.54 lowered titers when the infection was less than 3 months old. That was statistically significant compared to sera from patients with infections of greater than 6 months (mean 0.20 ± 0.41) and with infections between 3 and 6 months (mean, 1.17 ± 0.41) ( P = 0.0022 and P < 0.0001, respectively). These results were visualized by Western blotting. We concluded that high avidity (≤1 lowered titer) ruled out infection during the last 6 months and that complete denaturation was related to an infection which had occurred within the previous 3 months. Between these two situations, the avidity test is inconclusive. We suggest using an avidity test for atypical Q fever serology that could be misclassified as residual antibodies (IgG against C. burnetii detected without active or recent infection) and for pregnant women risking obstetrical complications. This new test will dramatically improve the diagnosis and management of patients with Q fever., (Copyright © 2019 American Society for Microbiology.)

Infection with Coxiella burnetii , the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA class II C. burnetii epitopes, providing the basis for a novel T-cell targeted subunit vaccine. In this study, we investigated in a cohort of 22 individuals treated for persistent infection (chronic Q fever) whether they recognize the same set of epitopes or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection. In cultured enzyme-linked immunosorbent spot (ELISpot) assays, individuals with chronic Q fever showed strong class II epitope-specific responses that were largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or nonreactive in subjects with chronic Q fever. Consistent with more recent/prolonged exposure, we found, however, stronger ex vivo responses by direct ELISpot to both whole-cell C. burnetii and individual peptides in chronic patients than in convalescents. In conclusion, we have validated and expanded a previously published set of candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in treated patients with chronic Q fever and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrated that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes., (Copyright © 2019 American Society for Microbiology.)

Background: Brucellosis, coxiellosis, and toxoplasmosis can be transmitted from infected ruminants to pregnant women and may induce adverse pregnancy outcomes; however, there are to date few studies. This study aimed to examine the seropositivities of immunoglobulin G (IgG) against those three pathogens among pregnant women with adverse pregnancy outcomes, and to explore the associated factors., Methods: A cross-sectional study was conducted in southern Thailand, where goat production is common. A total of 105 pregnant Thai women who had adverse pregnancy outcomes and serum samples collected at first antenatal care visit before their 28th gestational week from June 2015 to June 2016 were included. The seropositivities of IgG anti-Brucella abortus, Toxoplasma gondii, and Coxiella burnetii antibodies were tested by using commercial enzyme-linked immunosorbent assay (ELISA) kits. Associated factors with seropositivity were analyzed using multiple logistic regression., Results: Most women were Muslim aged 20-34 years and 32.4% had a prior history of one or more adverse pregnancy outcomes. One-third of the women had been exposed to goats or raw goat products. Of the 105 serum samples, the seropositivity of anti-T. gondii IgG was highest (33/105, 31.4%), followed by anti-C. burnetii IgG (2/105, 1.9%), and anti-B. abortus IgG (1/105, 1.0%), respectively. None of the pregnant women were found to be co-seropositive for those three pathogens., Conclusions: One-third of women with adverse pregnancy outcomes showed positive antibodies for toxoplasmosis, coxiellosis and brucellosis. A dose-response relationship between seropositivity of anti-T. gondii IgG and age was noticed., Competing Interests: The authors have declared that no competing interests exist.

Human Q fever is caused by the intracellular bacterial pathogen Coxiella burnetii Q fever presents with acute flu-like and pulmonary symptoms or can progress to chronic, severe endocarditis. After human inhalation, C. burnetii is engulfed by alveolar macrophages and transits through the phagolysosomal maturation pathway, resisting the acidic pH of lysosomes to form a parasitophorous vacuole (PV) in which to replicate. Previous studies showed that C. burnetii replicates efficiently in primary human alveolar macrophages (hAMs) in ex vivo human lung tissue. Although C. burnetii replicates in most cell types in vitro , the pathogen does not grow in non-hAM cells of human lung tissue. In this study, we investigated the interaction between C. burnetii and other pulmonary cell types apart from the lung environment. C. burnetii formed a prototypical PV and replicated efficiently in human pulmonary fibroblasts and in airway, but not alveolar, epithelial cells. Atypical PV expansion in alveolar epithelial cells was attributed in part to defective recruitment of autophagy-related proteins. Further assessment of the C. burnetii growth niche showed that macrophages mounted a robust interleukin 8 (IL-8), neutrophil-attracting response to C. burnetii and ultimately shifted to an M2-polarized phenotype characteristic of anti-inflammatory macrophages. Considering our findings together, this study provides further clarity on the unique C. burnetii -lung dynamic during early stages of human acute Q fever., (Copyright © 2019 American Society for Microbiology.)

In hypoxic and inflamed tissues, oxygen (O 2 )-dependent antimicrobial defenses are impaired due to a shortage of O 2 . To gain insight into the mechanisms that control bacterial infection under hypoxic conditions, we infected macrophages with the obligate intracellular pathogen Coxiella burnetii, the causative agent of Q fever. Our experiments revealed that hypoxia impeded C. burnetii replication in a hypoxia-inducible factor (HIF) 1α-dependent manner. Mechanistically, under hypoxia, HIF1α impaired the activity of STAT3, which in turn reduced the intracellular level of TCA cycle intermediates, including citrate, and impeded C. burnetii replication in macrophages. However, bacterial viability was maintained, allowing the persistence of C. burnetii, which is a prerequisite for the development of chronic Q fever. This knowledge will open future research avenues on the pathogenesis of chronic Q fever. In addition, the regulation of TCA cycle metabolites by HIF1α represents a previously unappreciated mechanism of host defense against intracellular pathogens., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Coxiella burnetii , the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select C. burnetii epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine. Immunoinformatic methods were used to predict 65 HLA class I epitopes and 50 promiscuous HLA class II C. burnetii epitope clusters, which are conserved across strains of C. burnetii . HLA binding assays confirmed 89% of class I and 75% of class II predictions, and 11 HLA class II epitopes elicited IFNγ responses following heterologous DNA/DNA/peptide/peptide prime-boost immunizations of HLA-DR3 transgenic mice. Human immune responses to the predicted epitopes were characterized in individuals naturally exposed to C. burnetii during the 2007-2010 Dutch Q fever outbreak. Subjects were divided into three groups: controls with no immunological evidence of previous infection and individuals with responses to heat-killed C. burnetii in a whole blood IFNγ release assay (IGRA) who remained asymptomatic or who experienced clinical Q fever during the outbreak. Recall responses to C. burnetii epitopes were assessed by cultured IFNγ ELISpot. While HLA class I epitope responses were sparse in this cohort, we identified 21 HLA class II epitopes that recalled T-cell IFNγ responses in 10-28% of IGRA+ subjects. IGRA+ individuals with past asymptomatic and symptomatic C. burnetii infection showed a comparable response pattern and cumulative peptide response which correlated with IGRA responses. None of the peptides elicited reactogenicity in a C. burnetii exposure-primed guinea pig model. These data demonstrate that a substantial proportion of immunoinformatically identified HLA class II epitopes show long-lived immunoreactivity in naturally infected individuals, making them desirable candidates for a novel human multi-epitope Q fever vaccine.

Laboratory testing for Q fever (Coxiella burnetii) is essential for a differential diagnosis, yet little is known about Q fever diagnostic testing practices in the United States. We retrospectively analyzed Q fever immunoglobulin G (IgG) indirect immunofluorescence assay (IFA) testing data between 1/1/2012-10/31/2016 from ARUP, LabCorp, Mayo Medical Laboratories, and Quest Diagnostics. Data included IgG phase I and phase II titers, patient age and sex, and state and date of specimen collection. On average, 12,821 specimens were tested for Q fever annually by the participating laboratories. Of 64,106 total specimens, 84.1% tested negative for C. burnetii-specific antibodies. Positive titers ranged from 16 to 262,144 against both phase I and phase II antigens. Submission of specimens peaked during the summer months, and more specimens were submitted from the West North Central division. Testing occurred more frequently in males (53%) and increased with age. In conclusion, few U.S. Q fever cases are reported, despite large volumes of diagnostic specimens tested. Review of commercial laboratory data revealed a lack of paired serology samples and patterns of serology titers that differ from case reporting diagnostic criteria.

Cytokine responses of chronic Q fever patients to the intracellular bacterium Coxiella burnetii have mostly been studied using ex vivo stimulation of immune cells with heat-killed C. burnetii due to the extensive measures needed to work with viable biosafety level 3 agents. Whether research with heat-killed C. burnetii can be translated to immune responses to viable C. burnetii is imperative for the interpretation of previous and future studies with heat-killed C. burnetii Peripheral blood mononuclear cells (PBMCs) of chronic Q fever patients ( n = 10) and healthy controls ( n = 10) were stimulated with heat-killed or viable C. burnetii of two strains, Nine Mile and the Dutch outbreak strain 3262, for 24 h, 48 h, and 7 days in the absence or presence of serum containing anti- C. burnetii antibodies. When stimulated with viable C. burnetii , PBMCs of chronic Q fever patients and controls produced fewer proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-1β) after 24 h than after stimulation with heat-killed C. burnetii In the presence of Q fever seronegative serum, IL-10 production was higher after stimulation with viable rather than heat-killed C. burnetii ; however, when incubating with anti- C. burnetii antibody serum, the effect on IL-10 production was reduced. Levels of adaptive, merely T-cell-derived cytokine (gamma interferon, IL-17, and IL-22) and CXCL9 production were not different between heat-killed and viable C. burnetii stimulatory conditions. Results from previous and future research with heat-killed C. burnetii should be interpreted with caution for innate cytokines, but heat-killed C. burnetii -induced adaptive cytokine production is representative of stimulation with viable bacteria., (Copyright © 2018 American Society for Microbiology.)

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a , Il1b , and Tnfa , the chemokine genes Cxcl2 and Ccl5 , and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response., (Copyright © 2018 American Society for Microbiology.)

A pilot seroprevalence study was performed among asymptomatic occupationally exposed individuals in June, 2016 in the Peace River region of Alberta and British Columbia. Five of 40 subjects - 3 of 24 small ruminant producers, 1 of 14 abattoir workers, and 1 of 2 veterinarians had evidence of Coxiella exposure. More systematic surveillance and more active promotion of biosecure husbandry methods should be considered.

Q fever is a zoonotic disease caused by Coxiella burnetii that usually presents with non-specific or benign constitutional symptoms. Diagnosis is often challenging and, after acute Q fever, 1%-5% of patients can develop chronic disease. We present an 80-year-old male patient who was admitted due to a 3 months history of fever, productive cough, myalgia, weight loss, headache and hearing loss. Chronic Q fever was confirmed by positive antiphase I immunoglobulin G. Frequent locations of chronic infection was discarded, and ear CT revealed a right mastoid infection. He was treated with doxycycline and hydroxychloroquine for 18 months with significant improvement. This is a rare case of chronic Q fever presenting with otomastoiditis that has never been described., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Our previous study demonstrated that neutrophils play an important role in host defense against Coxiella burnetii infection in mice. In this study, avirulent strain C. burnetii Nine Mile phase II (NMII) was used to examine if C. burnetii can modulate mouse bone marrow-derived neutrophil apoptosis. The results indicated that NMII can inhibit neutrophil apoptosis. Western blotting demonstrated that caspase-3 cleavage was decreased in NMII-infected neutrophils, while phosphorylated mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase 1 (Erk1) were increased. Additionally, p38, Erk1/2, phosphoinositide 3-kinase (PI3K), or NF-κB inhibitors reduced the ability of NMII to inhibit neutrophil apoptosis. These results suggest that NMII-mediated inhibition of neutrophil apoptosis depends on its ability to activate neutrophil MAPK pathways. Antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) was significantly increased in NMII-infected neutrophils, and an Mcl-1 inhibitor significantly reduced the ability of NMII to inhibit neutrophil apoptosis. Mcl-1 protein stability was enhanced by phosphorylation at Thr-163 by Erk, and the protein levels were regulated by p38, Erk, PI3K, and NF-κB. Furthermore, the observation that a type IV secretion system (T4SS)-deficient dotA mutant showed a significantly reduced ability to inhibit neutrophil apoptosis compared to wild-type (WT) NMII suggests that T4SS-secreted factors may be involved in NMII-induced inhibition of neutrophil apoptosis. Collectively, these results demonstrate that NMII inhibits neutrophil apoptosis through inhibition of caspase-3 cleavage and activation of MAPK survival pathways with subsequent expression and stabilization of antiapoptotic protein Mcl-1, a process that may partially require the T4SS., (Copyright © 2018 American Society for Microbiology.)

To understand the role of class I major histocompatibility complex (MHC-I) and class II MHC (MHC-II) antigen presentation pathways in host defense against Coxiella burnetii infection, we examined whether MHC-I or MHC-II deficiency in mice would significantly influence their susceptibility to virulent C. burnetii Nine Mile phase I (NMI) infection. The results indicate that NMI infection induced more severe disease in both MHC-I-deficient and MHC-II-deficient mice than in wild-type (WT) mice, while only MHC-I-deficient mice developed a severe persistent infection and were unable to control bacterial replication. These results suggest that both MHC-I-restricted CD8 + T cells and MHC-II-restricted CD4 + T cells contribute to host defense against primary C. burnetii infection, while MHC-I-restricted CD8 + T cells appear to play a more critical role in controlling bacterial replication. Additionally, although NMI infection induced more severe disease in TAP1-deficient mice than in their WT counterparts, TAP1 deficiency in mice did not significantly influence their ability to eliminate C. burnetii This suggests that C. burnetii antigen presentation to CD8 + T cells by the MHC-I classical pathway may depend only partially on TAP1. Furthermore, granzyme B deficiency in mice did not significantly alter their susceptibility to C. burnetii infection, but perforin-deficient mice were unable to control host inflammatory responses during primary C. burnetii infection. These results suggest that perforin, but not granzyme B, is required for C. burnetii antigen-specific cytotoxic CD8 + T cells to control primary C. burnetii infection., (Copyright © 2018 American Society for Microbiology.)

BackgroundAfter a large Q fever outbreak in the Netherlands in the period from 2007 to 2010, the risk of Q fever transmission through tissue and cell transplantation from undiagnosed chronic Q fever cases became a potential issue. Aim: We aimed to evaluate the risk of Q fever transmission through tissue and cell transplantation. Methods: We performed a retrospective observational cohort study among 15,133 Dutch donors of tissues and stem cells from 2010 to 2015 to assess seroprevalence of Coxiella burnetii antibodies, to identify factors associated with presence of C. burnetii antibodies, and to assess the proportion of undiagnosed chronic Q fever cases. Results: The study population consisted of 9,478 (63%) femoral head donors, 5,090 (34%) post-mortal tissue donors and 565 (4%) cord blood donors. Seroprevalence of C. burnetii antibodies gradually decreased after the outbreak, from 2.1% in 2010 to 1.4% in 2015, with a significant trend in time (p < 0.001). Of 301 seropositive donors, seven (2.3%) were newly detected with chronic Q fever (0.05% of all screened donors). Conclusion: This study shows that seroprevalence of C. burnetii antibodies among donors of tissues and cells in the Netherlands after 2014 was similar to pre-outbreak levels in the general population. The proportion of newly detected chronic Q fever patients among donors of tissues and cells was smaller than 0.1%. This study may prompt discussion on when to terminate the screening programme for chronic Q fever in donors of tissues and cells in the Netherlands.

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Clinical presentation in humans varies from asymptomatic to flu-like illness and severe sequelae may be seen. Ruminants are often sub-clinically infected or show reproductive disorders such as abortions. In Egypt, only limited data on the epidemiology of Q fever in animals are available. Using a stratified two stage random sampling approach, we evaluated the prevalence of Coxiella burnetii specific antibodies among ruminants and camels in 299 herds. A total of 2,699 blood samples was investigated using enzyme-linked-immunosorbent assay (ELISA). Coxiella burnetii specific antibodies were detected in 40.7% of camels (215/528), 19.3% of cattle (162/840), 11.2% of buffaloes (34/304), 8.9% of sheep (64/716) and 6.8% of goats (21/311), respectively. Odds of seropositivity were significantly higher for cattle (aOR: 3.17; 95% CI: 1.96-5.13) and camels (aOR: 9.75; 95% CI: 6.02-15.78). Significant differences in seropositivity were also found between domains (Western Desert, Eastern Desert and Nile Valley and Delta) and 25 governorates (p < 0.001), respectively. Animal rearing in the Eastern Desert domain was found to be a significant risk factor (aOR: 2.16; 95% CI: 1.62-2.88). Most seropositive animals were older than four years. No correlation between positive titers and husbandry practices or animal origin were found (p > 0.05). Only 8.7% of the interviewed people living on the farms consumed raw camel milk and none reported prior knowledge on Q fever. Findings from this nationwide study show that exposure to Coxiella burnetii is common in ruminants and camels. Disease awareness among physicians, veterinarians and animal owners has to be raised. Future epidemiological investigations have to elucidate the impact of Q fever on human health and on the economy of Egypt.

Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella ( C .) burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM) represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. S trains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III) to high (MLVA group IV). Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80) on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

Introduction: Coxiella burnetii is an obligatory intracellular bacterial pathogen causing the zoonotic disease Q fever. The most common reservoirs of C. burnetii  are wild mammals, birds and ticks. Pregnant domestic ruminants infected with this bacterium are also a major source of human infection., Material and Methods: The serological prevalence of C. burnetii in goats and sheep diagnosed with adverse pregnancy outcomes was assessed by undertaking a survey on 800 dairy goats and 800 dairy ewes reared in four different regions of Greece (Macedonia, Thrace, Thessaly, and Peloponnese). A stratified sampling was carried out, taking also as a criterion the age of the animals. Serum antibodies were analyzed by a commercial ELISA according to the manufacturer's recommendations., Results: Generally, there was a statistically significantly higher serological prevalence of C. burnetii (14.4%) in goats compared to sheep (8%). Serological prevalence was higher in adults (15.5% in goats and 8.5% in sheep) compared to yearlings (7.4% in goats and 4.6% in sheep). The prevalence increased significantly with age only in goats. Finally, all animals reared in Peloponnese had a prevalence significantly higher (21% in goats and 18% in sheep) than animals reared in the other three regions., Conclusions: To the best of the authors' knowledge, this is the first report that associates C. burnetii with reproductive disturbances of domestic ruminants in Greece. However, considering the importance of coxiellosis for public health, further investigations are required on its epidemiology regarding abortion, premature delivery, stillbirth and weak offspring in small ruminants, as well as in other domestic and wild animal species.

A small gram-negative bacterium, Coxiella burnetii ( C. burnetii ), is responsible for a zoonosis called Q fever. C. burnetii is an intracellular bacterium that can survive inside microbicidal cells like monocytes and macrophages by hijacking several functions of the immune system. Among several virulence factors, the lipopolysaccharide (LPS) of C. burnetii is one of the major factors involved in this immune hijacking because of its atypical composition and structure. Thus, the aim of this mini-review is to summarize the repressive effects of C. burnetii LPS on the antibacterial immunity of cells., Competing Interests: The authors declare no conflict of interest.

Background: Prior to the 2007-2010 Q fever epidemic in the Netherlands, the seroprevalence of antibodies against Coxiella burnetii in the general population was 1.5%, which is low compared to other countries. We aimed to determine the seroprevalence after the Q fever epidemic among people living in the affected area, compare the seroprevalence with the incidence of Q fever notifications during the 2007-2010 Q fever epidemic, and to identify farm exposures associated with having antibodies against C. burnetii., Methods: During the period March 2014-February 2015, residents aged 18-70 years from two provinces were invited by general practitioners to complete a questionnaire on their symptoms and personal characteristics and to submit a blood sample. We used the mandatory provincial database of livestock licences to calculate distance to farms/farm animals for each participant. To compare ELISA-positive participants for C. burnetii antibodies with those who were negative, we calculated prevalence ratios (PR) using binominal regression. We compared the C. burnetii seroprevalence in the period March 2014-February 2015 with the incidence of Q fever notifications during the 2007-2010 Q fever epidemic at municipal level by calculating the Spearman correlation coefficient., Results: Of the 2296 participants (response rate: 34%), 6.1% (n = 139, 95% CI 5.1-7.1%) had C. burnetii antibodies (range in municipalities: 1.7-14.1%). C. burnetii seroprevalence was higher in individuals living within 1000 m of goat farms (PR 3.0; 95% CI 1.4-6.4) or within 1000 m of > 50 goats (PR 1.9; 95% CI 1.2-3.0). Seroprevalence increased with decreasing distance to the closest goat farm that was infected during the epidemic years (< 500 m, PR 9.5, 95% CI 2.8-32; 500-1000 m, PR 4.5, 95% CI 2.6-7.7; 1000-1500 m, PR 2.2, 95% CI 1.1-4.3, 1500-2000 m, PR 1.2, 95% CI 0.6-2.5; > 2000 reference group). There was no significant correlation between C. burnetii seroprevalence and Q fever incidence during the 2007-2010 epidemic (r s  = 0.42, p = 0.156)., Conclusions: Results showed a remarkable spatial variation in C. burnetii seroprevalence in a relatively small livestock dense area. It confirms previous evidence that the Q fever epidemic was primarily the result of airborne C. burnetii transmission from Q fever affected goat farms.

Vaccination against Coxiella burnetii, the cause of Q fever, is reportedly the only feasible strategy of eradicating infection in ruminant herds. Preventive vaccination of seronegative goats is more effective in reducing shedding of C. burnetii than vaccinating seropositive goats. The age at which goats born on heavily-contaminated farms first seroconvert to C. burnetii has not yet been documented. In a 16-month birth cohort study, the age at which goats seroconverted against C. burnetii was investigated; 95 goats were bled every 2 weeks and tested for antibodies against C. burnetii. Risk factors for seroconversion were explored and goats shedding C. burnetii were identified by testing vaginal swabs taken at the goats' first kidding using a com1 polymerase chain reaction assay. The first surge in the number of goats with IgM to C. burnetii was observed at week 9. Thus, a first vaccination not later than 8 weeks of age to control C. burnetii in highly contaminated environments is indicated. The odds of seroconversion were 2.0 times higher [95% confidence interval (CI) 1.2, 3.5] in kids born by does with serological evidence of recent infection (IgM seropositive) compared to kids born by IgM seronegative does, suggesting either in utero transmission or peri-parturient infection. The rate of seroconversion was 4.5 times higher (95% CI 2.1, 9.8) during than outside the kidding season, highlighting the risk posed by C. burnetii shed during kidding, even to goats outside the kidding herd. Shedding of C. burnetii at kidding was detected in 15 out of 41 goats infected before breeding.

Background & Objectives: Seroprevalence of Q fever (QF) caused by Coxiella burnetii has been reported from different parts of India. Usually serological/molecular tests are employed for detection of infection. The present study was undertaken to verify the validity of three different QF phase II IgM ELISA kits for acute QF diagnosis by comparing with the gold standard indirect fluorescent antibody assay (IFA)., Methods: Fifty eight serum samples collected from 42 patients (26 patients provided acute sample only and 16 both acute and convalescent samples) which were examined by all three commercial kits, were cross-checked with QF Phase II IgM IFA for confirmation., Results: Eleven patients were positive for C. burnetii antibodies by IFA in acute and/or convalescent serum samples. Taking IFA as a reference, percentages of sensitivity, specificity, positive predictive value and negative predictive value for Virion-Serion/Vircell/NovaTec were 36.36, 61.29, 25.00, 73.08; 81.82, 35.48, 31.03, 84.62 and 100, 25.81, 32.35, 100 per cent, respectively., Interpretation & Conclusions: The three different ELISA kits exhibited poor agreement amongst them and unacceptable level of false positivity. IFA remains to be the only option for diagnosing acute QF. Discrepancy between the clinical findings and IFA/ELISA results needs confirmation by C. burnetii DNA detection in real-time polymerase chain reaction.

Q fever is a neglected and potentially fatal disease. During acute Q fever, antiphospholipid antibodies are very prevalent and have been associated with fever, thrombocytopenia, acquired heart valve disease, and progression to chronic endocarditis. However, thrombosis, the main clinical criterion of the 2006 updated classification of the antiphospholipid syndrome, has not been assessed in this context. To test whether thrombosis is associated with antiphospholipid antibodies and whether the criteria for antiphospholipid syndrome can be met in patients with acute Q fever, we conducted a cross-sectional study at the French National Referral Center for Q fever.Patients included were diagnosed with acute Q fever in our Center between January 2007 and December 2015. Each patient's history and clinical characteristics were recorded with a standardized questionnaire. Predictive factors associated with thrombosis were assessed using a rare events logistic regression model. IgG anticardiolipin antibodies (IgG aCL) assessed by an enzyme-linked immunosorbent assay were tested on the Q fever diagnostic serum. A dose-dependent relationship between IgG aCL levels and thrombosis was tested using a receiver operating characteristic (ROC) analysis.Of the 664 patients identified for inclusion in the study, 313 (47.1%) had positive IgG aCL and 13 (1.9%) were diagnosed with thrombosis. Three patients fulfilled the antiphospholipid syndrome criteria. After multiple adjustments, only positive IgG aCL (relative risk, 14.46 [1.85-113.14], P = .011) were independently associated with thrombosis. ROC analysis identified a dose-dependent relationship between IgG aCL levels and occurrence of thrombosis (area under curve, 0.83, 95%CI [0.73-0.93], P < .001).During acute Q fever, antiphospholipid antibodies are associated with thrombosis, thrombocytopenia, and acquired valvular heart disease. Antiphospholipid antibodies should be systematically assessed in acute Q fever patients. Hydroxychloroquine, which has been previously shown to antagonize IgG aCL pathogenic properties, should be tested in acute Q fever patients with anticardiolipin antibodies to prevent antiphospholipid-associated complications.Key Point: In addition to fever, thrombocytopenia and acquired valvular heart disease, antiphospholipid antibodies are associated with thrombosis during acute Q fever.

Coxiella burnetii is the causative agent of Q fever, a zoonotic disease that threatens both human and animal health. Due to the paucity of experimental animal models, little is known about how host factors interface with bacterial components and affect pathogenesis. Here, we used Drosophila melanogaster , in conjunction with the biosafety level 2 (BSL2) Nine Mile phase II (NMII) clone 4 strain of C. burnetii , as a model to investigate host and bacterial components implicated in infection. We demonstrate that adult Drosophila flies are susceptible to C. burnetii NMII infection and that this bacterial strain, which activates the immune deficiency (IMD) pathway, is able to replicate and cause mortality in the animals. We show that in the absence of Eiger, the only known tumor necrosis factor (TNF) superfamily homolog in Drosophila , Coxiella -infected flies exhibit reduced mortality from infection. We also demonstrate that the Coxiella type 4 secretion system (T4SS) is critical for the formation of the Coxiella -containing vacuole and establishment of infection in Drosophila Altogether, our data reveal that the Drosophila TNF homolog Eiger and the Coxiella T4SS are implicated in the pathogenesis of C. burnetii in flies. The Drosophila /NMII model mimics relevant aspects of the infection in mammals, such as a critical role of host TNF and the bacterial T4SS in pathogenesis. Our work also demonstrates the usefulness of this BSL2 model to investigate both host and Coxiella components implicated in infection., (Copyright © 2017 American Society for Microbiology.)

AbstractLittle is known about the burden of Q fever in Thailand. We conducted a serological study to describe the prevalence of anti- Coxiella burnetii antibodies among ruminants and occupationally exposed persons in response to the report of the first two Q fever endocarditis patients in Thailand in 2012. We randomly selected ruminant sera from brucellosis surveillance and examined sera of 661 occupationally exposed subjects from two provinces of Thailand: Chiangmai and Nakornratchasima. Animal and human sera were tested using commercial enzyme-linked immunosorbent assay (ELISA). Environmental samples, vaginal swab, and milk from cows in Chiangmai farms with detectable anti- C. burnetii serum antibodies were tested using polymerase chain reaction (PCR). Among the 1,632 animal sera tested, 64 (3.9%) were seropositive. The prevalence was highest in dairy cattle (4.6%, 45/988), followed by goats (3.5%, 18/516) and sheep (2.1%, 1/48). The prevalence of anti- C. burnetii antibodies in each species varied significantly by province: the prevalence in cattle was higher in Chiangmai (5.5% versus 0%), however, the prevalence in sheep and goats was higher in Nakornratchasima (5.9% versus 1.0%). Four out of 60 milk samples were positive by PCR (6.7%). No environmental samples were positive. Among 661 human samples, 83 (12.6%) were ELISA positive. Seroprevalence was statistically higher in Chiangmai compare with Nakornratchasima (42.8% versus 3.0%). Coxiella burnetii infection exists in Thailand, but the prevalence varies by geographic distribution and animal reservoirs. Further studies focusing on the burden and risk factors of C. burnetii infection among high-risk groups should be conducted.

Objectives: Q fever is caused by Coxiella burnetii, an intracellular bacterium that infects phagocytes. The aim of the present study was to investigate whether the C. burnetii-induced IFN-γ response is defective in chronic Q fever patients., Methods: IFN-γ was measured in supernatants of C. burnetii-stimulated peripheral blood mononuclear cells (PBMCs) of 17 chronic Q fever patients and 17 healthy individuals. To assess IFN-γ responses, expression profiles of IFN-γ-induced genes in C. burnetii-stimulated PBMCs were studied in six patients and four healthy individuals. Neopterin was measured in PBMC supernatants (of eight patients and four healthy individuals) and in sera (of 21 patients and 11 healthy individuals). In a genetic association study, polymorphisms in genes involved in the Th1-cytokine response were analysed in a cohort of 139 chronic Q fever patients and a cohort of 220 control individuals with previous exposition to C. burnetii., Results: IFN-γ production by C. burnetii-stimulated PBMCs from chronic Q fever patients was significantly higher than in healthy controls. Many IFN-γ response genes were strongly upregulated in PBMCs of patients. Neopterin levels were significantly higher in PBMC supernatants and sera of patients. The IL12B polymorphisms rs3212227 and rs2853694 were associated with chronic Q fever., Conclusions: IFN-γ production, as well as the response to IFN-γ, is intact in chronic Q fever patients, and even higher than in healthy individuals. Polymorphisms in the IL-12p40 gene are associated with chronic Q fever. Thus, a deficiency in IFN-γ responses does not explain the failure to clear the infection. The genetic data suggest, however, that the IL-12/IFN-γ pathway does play a role., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: Q fever is a zoonotic disease that occurs worldwide; however, little is known about its prevalence in South Korea. We attempted to determine the prevalence of Q fever seroreactivity among Korean slaughterhouse workers and the risk factors for seroreactivity according to the type of work., Methods: The study was conducted among 1503 workers at a total of 73 slaughterhouses and 62 residual-product disposal plants. During the study period, sites were visited and surveys were administered to employees involved in slaughterhouse work, and serological tests were performed on blood samples by indirect immunofluorescence assays. Serological samples were grouped by job classification into those of slaughter workers, residual-product handlers, inspectors and inspection assistants, and grading testers and testing assistants. Employee risk factors were analyzed according to the type of work., Results: Out of 1481 study subjects who provided a blood sample, 151 (10.2%) showed reactive antibodies. When these results were analyzed in accordance with the type of work, the result of slaughter workers (11.3%) was similar to the result of residual-product handlers (11.4%), and the result of inspectors and assistants (5.3%) was similar to the result of grading testers and assistants (5.4%). Among those who answered in the affirmative to the survey question, "Has there been frequent contact between cattle blood and your mouth while working?" the proportions were 13.4 and 4.6%, respectively, and this was identified as a risk factor that significantly varied between job categories among slaughterhouse workers., Conclusions: This study found a Q fever seroreactivity rate of 10.2% for slaughterhouse workers, who are known to be a high-risk population. Contact with cattle blood around the mouth while working was the differential risk factor between job categories among slaughterhouse workers.