Coxiella burnetii inhibits host immunity by a protein phosphatase adapted from glycolysis.

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.

The Feasibility of Using Coxiella burnetii Avirulent Nine Mile Phase II Viable Bacteria as a Live Attenuated Vaccine Against Q fever.

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.

Molecular diagnosis of Coxiella burnetii in culture negative endocarditis and vascular infection in South Korea.

BACKGROUND: Q fever endocarditis is a major cause of culture-negative endocarditis. The role of Coxellia burnetii is underestimated because it is difficult to diagnose. We investigated the significance of C. burnetii as the cause of culture-negative endocarditis and vascular infection by examining blood and tissue specimens using serological testing and polymerase chain reaction (PCR). METHODS: All patients with infective endocarditis or large vessel vasculitis were prospectively enrolled at a tertiary-care hospital from May 2016 through September 2020. Q fever endocarditis and vascular infection were diagnosed based on: (1) positive PCR for a cardiac valve or vascular tissue, (2) positive PCR for blood or phase I immunoglobulin G (IgG) ≥ 6400, or (3) phase I IgG ≥ 800 and < 6400 with morphologic abnormality. PCR targeted C. burnetii transposase gene insertion element IS1111a. RESULTS: Of the 163 patients, 40 (25%) had culture-negative endocarditis (n = 35) or vascular infection (n = 5). Of the 40 patients, 24 (60%) were enrolled. Eight (33%) were diagnosed with Q fever endocarditis or vascular infection. Of these 8 patients, 6 had suspected acute Q fever endocarditis or vascular infection with negative phase I IgG. Six patients were not treated for C. burnetii, 4 were stable after surgery. One patient died due to surgical site infection after 5 months post-operatively and one died due to worsening underlying disease. CONCLUSIONS: Approximately one-third of patients with culture-negative endocarditis and vascular infection was diagnosed as Q fever. Q fever endocarditis and vascular infection may be underestimated in routine clinical practice in South Korea.KEY MESSAGEQ fever endocarditis and vascular infection may be underestimated in routine clinical practice, thus, try to find evidence of C. burnetti infection in suspected patients by all available diagnostic tests including PCR.

Whole Blood Interferon γ Release Is a More Sensitive Marker of Prior Exposure to Coxiella burnetii Than Are Antibody Responses.

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.

SEROLOGICAL EVIDENCE OF COXIELLA BURNETII INFECTION IN THE WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN SOUTH AFRICA.

Coxiellosis, or Query (Q) fever, a disease caused by the intracellular bacteria Coxiella burnetii, was recently described in a managed breeding herd of white rhinoceros (Ceratotherium simum) in the southeastern United States. Clinical disease often results in abortion and could represent a conservation challenge for this species. In addition to the reproductive and herd management consequences, coxiellosis is also a zoonotic disease. Infection or clinical disease in any free-ranging rhinoceros species in a national park setting has not been previously described. In this study, evidence of prior infection was measured by immunofluorescent antibody titers in 89 serum samples collected from white rhinoceros within private reserves and a national park in South Africa. Total seropositivity was 48/89 (53.9% [95% CI, 43.6-63.9%]). Animals on private reserves had a seropositivity of 21/51 (41.1% [95% CI, 27.1-55.2%]), and national park rhinoceros had a higher rate of seropositivity at 71.0% [95% CI, 55.9-86.2%] (27/38; P= 0.004). Adults had a higher seropositivity compared with subadults (P= 0.03). There was no difference in seropositivity between sexes (P > 0.05). Results demonstrate that South African white rhinoceros populations are exposed to Coxiella, which could result in underrecognized reproductive consequences. Further studies should investigate potential implications for public health and conservation management of this species.

Subunit Vaccines Using TLR Triagonist Combination Adjuvants Provide Protection Against Coxiella burnetii While Minimizing Reactogenic Responses.

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.

Serological evidence of single and mixed infections of Rift Valley fever virus, Brucella spp. and Coxiella burnetii in dromedary camels in Kenya.

Camels are increasingly becoming the livestock of choice for pastoralists reeling from effects of climate change in semi-arid and arid parts of Kenya. As the population of camels rises, better understanding of their role in the epidemiology of zoonotic diseases in Kenya is a public health priority. Rift Valley fever (RVF), brucellosis and Q fever are three of the top priority diseases in the country but the involvement of camels in the transmission dynamics of these diseases is poorly understood. We analyzed 120 camel serum samples from northern Kenya to establish seropositivity rates of the three pathogens and to characterize the infecting Brucella species using molecular assays. We found seropositivity of 24.2% (95% confidence interval [CI]: 16.5-31.8%) for Brucella, 20.8% (95% CI: 13.6-28.1%) and 14.2% (95% CI: 7.9-20.4%) for Coxiella burnetii and Rift valley fever virus respectively. We found 27.5% (95% CI: 19.5-35.5%) of the animals were seropositive for at least one pathogen and 13.3% (95% CI: 7.2-19.4%) were seropositive for at least two pathogens. B. melitensis was the only Brucella spp. detected. The high sero-positivity rates are indicative of the endemicity of these pathogens among camel populations and the possible role the species has in the epidemiology of zoonotic diseases. Considering the strong association between human infection and contact with livestock for most zoonotic infections in Kenya, there is immediate need to conduct further research to determine the role of camels in transmission of these zoonoses to other livestock species and humans. This information will be useful for designing more effective surveillance systems and intervention measures.

Take my breath away: studying pathogen invasion of the human lung using primary tissue models.

The human pulmonary environment is complex, containing a matrix of cells, including fibroblasts, epithelial cells, interstitial macrophages, alveolar macrophages and neutrophils. When confronted with foreign material or invading pathogens, these cells mount a robust response. Nevertheless, many bacterial pathogens with an intracellular lifecycle stage exploit this environment for replication and survival. These include, but are not limited to, Coxiella burnetii, Legionella pneumophila, Yersinia pestis, Mycobacterium tuberculosis and Staphylococcus aureus. Currently, few human disease-relevant model systems exist for studying host-pathogen interactions during these bacterial infections in the lung. Here, we present two novel infection platforms, human alveolar macrophages (hAMs) and human precision-cut lung slices (hPCLS), along with an up-to-date synopsis of research using said models. Additionally, alternative uses for these systems in the absence of pathogen involvement are presented, such as tissue banking and further characterization of the human lung environment. Overall, hAMs and hPCLS allow novel human disease-relevant investigations that other models, such as cell lines and animal models, cannot completely provide.

Prevalence of Coxiella burnetii seropositivity and shedding in farm, pet and feral cats and associated risk factors in farm cats in Quebec, Canada.

Cats represent a potential source of Coxiella burnetii, the aetiological agent of Q fever in humans. The prevalence and risk factors of C. burnetii infection in farm, pet and feral cats were studied in Quebec, Canada, using a cross-sectional study. Serum samples were tested using a specific enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against C. burnetii, whereas rectal swabs were assayed using real-time quantitative polymerase chain reaction (qPCR) for the molecular detection of the bacteria. Potential risk factors for farm cats were investigated using clinical examinations, questionnaires and results from a concurrent study on C. burnetii farm status. A total of 184 cats were tested: 59 from ruminant farms, 73 pets and 52 feral cats. Among farm cats, 2/59 (3.4%) were ELISA-positive, 3/59 (5.1%) were ELISA-doubtful and 1/59 (1.7%) was qPCR-positive. All pets and feral cats were negative to C. burnetii ELISA and qPCR. Farm cat positivity was associated with a positive C. burnetii status on the ruminant farm (prevalence ratio = 7.6, P = 0.03). Our results suggest that although pet and feral cats do not seem to pose a great C. burnetii risk to public health, more active care should be taken when in contact with cats from ruminant farms.

Prevalence and spatial distribution of Coxiella burnetii seropositivity in northern Australian beef cattle adjusted for diagnostic test uncertainty.

Q fever is a zoonotic disease caused by infection with Coxiella burnetii transmitted from animals including, but not limited to, cattle, sheep and goats. The infection in cattle is typically sub-clinical with some evidence suggesting associated reproductive loss. There is currently limited data on the true prevalence and distribution of coxiellosis in beef cattle across northern Australia. During this study, 2,012 sera samples from beef cattle managed on commercial farms located in Queensland and the Northern Territory were tested using an indirect immunofluorescent assay (IFA) for serological evidence of IgG antibodies against C. burnetii. Bayesian latent class models were used to estimate the true prevalence, adjusted for diagnostic test sensitivity and specificity and incorporating the hierarchical structure of the cattle within farms and regions. In this study, cattle in the Northern Territory had lower estimated true prevalence than cattle within most regions of Queensland with the exception of south-east Queensland. Results from this study have described the geographic distribution and estimated the true prevalence of antibodies to C. burnetii in a sample of extensively managed beef cattle located across the tropical grazing regions of northern Australia.

Impact of Sex Hormones on Macrophage Responses to Coxiella burnetii.

Introduction: Q fever, a zoonosis caused by Coxiella burnetii, affects more males than females despite a similar level of exposure. A protective role of estradiol has been reported in mice, suggesting that sex hormones are involved in C. burnetii infection. We wondered whether the responses of monocytes and monocyte-derived macrophages (MDMs) to C. burnetii are influenced by sex hormones. Materials and Methods: The bacterial intracellular fate in monocytes was studied using quantitative PCR, and monocyte cytokine production in response to C. burnetii was assessed using qRT-PCR and immunoassays. Before infection, MDMs from males and females were incubated with testosterone and estradiol, respectively. Results: Bacterial uptake and persistence were similar in monocytes from males and females but were slightly increased in male MDMs. The expression of inflammatory genes, including those encoding TNF and CXCL10, was higher in MDMs from females than in MDMs from males infected by C. burnetii. Adding testosterone to male MDMs amplified their immunoregulatory properties, including increased expression of IL10 and TGFB genes and TGF-ß production in response to C. burnetii. In contrast, adding estradiol to MDMs from females had no effect on their inflammatory profile. Conclusion: The stronger inflammatory profile of macrophages from females may have a protective role, likely under estrogen control, while testosterone may affect disease progression by promoting an anti-inflammatory response. This finding may have consequences for personalized management of patients with Q fever.

Soluble antigens derived from Coxiella burnetii elicit protective immunity in three animal models without inducing hypersensitivity.

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.

Analysis of recombinant proteins for Q fever diagnostics.

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.

Case Report: Visceral Leishmaniasis Falsely Diagnosed as Q Fever.

Visceral leishmaniasis (VL) is a systemic infection caused by the protozoal parasite Leishmania, spread via the bloodstream to the reticuloendothelial system, through the bite of the sand fly. It is endemic in parts of Africa, South America, Asia, and Europe, including the Mediterranean. Here, we describe a case of VL that was initially diagnosed as Q fever based on positive Coxiella burnetii serology and showed a partial response to doxycycline treatment.

First Serological & Molecular Study of Coxiella burnetii in Stray, Domestic Cats, and Their Owners in Iran.

Coxiella burnetii, the agent of Q fever, is recognized as a worldwide zoonosis a wide host and potentially complex reservoir systems. Infected ruminants are the main source of infection for humans, but cats also represent a potential source of infection. The prevalence of C burnetii in cats in Iran is unknown and the risks of transmission to humans are undetermined. This study aimed to determine the prevalence of C burnetii in domestic cats and their owners. An Enzyme-linked immunosorbent assay was used for detection of anti-C burnetii antibodies in both cats and humans. Cats serum samples and humans serum samples (n = 85) were tested with indirect ELISA. C burnetii was diagnosed using real time- polymerase chain reaction. Antibodies were detected in 19 sera of 85 (22.35%) samples in stray cats, 9 sera of 78 (11.53%) samples of domestic cats and 4 sera of 78 (5.12%) samples of their owners. This first study of C burnetii prevalence in cats in Iran has indicated that positive samples can be found throughout the country and these results confirm that Iranian cats have been exposed to C burnetii. Moreover, this study demonstrates that cat owners, breeders and veterinary personnel might be at higher risk of exposure of C burnetii.

Q fever and prevention.

Coxiella burnetii is an intracellular, Gram-negative bacterium and the etiological agent of Q fever, a worldwide zoonotic disease with a considerable economic impact in the livestock industry. Domesticated ruminants like cattle, sheep, and goats are the main reservoirs of Q fever in men. Humans usually acquire the disease by inhaling contaminated aerosol produced by infected livestock. Acute infection is typically asymptomatic or manifests as a febrile flu-like illness or pneumonia. In 1-5% of primary infections, chronic Q fever may develop, which can be life-threatening and often presents as endocarditis. Q fever outbreaks usually occur from occupational exposure involving shepherds, veterinarians, animal handlers, abattoir or dairy workers, and laboratory personnel working with C. burnetii. Thus, prevention and control efforts should be primarily directed toward these groups and the surrounding environment. The most effective way of prophylaxis is vaccination. Although clinical data are lacking, vaccination should also be considered for persons who are at higher risk for development of chronic Q fever, including those with cardiac valve defects, vascular aneurysms, and immunocompromised patients. Therefore, development of an effective and harmless vaccine is a subject of constant interest of researchers for decades. This review summarizes the progress toward Q fever vaccine development.

Comparison of three serological tests for the detection of Coxiella burnetii specific antibodies in European wild rabbits.

BACKGROUND: Coxiella burnetii causes Q fever, a zoonotic bacterial disease with a multi-host cycle and reservoirs in wild and domestic animal species. Q fever has a significant impact on the Australian public health and economy but its ecology and contributing reservoir species remain poorly understood. In Europe, rabbits (Oryctolagus cuniculus) were identified as a major reservoir of C. burnetii and it is possible that they play a similar role in Australia. In absence of commercial kit available for rabbit, the Thermo Fisher - PrioCHECK™ Ruminant Q fever Ab Plate Kit was adapted to successfully screen rabbits population in Europe. However, this assay is not accessible in Australia and we assessed the equivalency of two commercially available kits in Australia - IDEXX - CHEKIT Q Fever Antibody ELISA kit and IDVet - ID Screen® Q Fever Indirect Multi-species with the Thermo Fisher kit (reference kit). RESULTS: A total of 94 rabbit sera were screened by all three ELISA kits using the same confirmed positive and negative controls. While the IDEXX kit failed to agree the other two assays (concordance correlation coefficient, rb < 0.77), IDVet kit showed satisfactory equivalency with Thermo Fisher (rb = 0.927). CONCLUSION: IDvet kit provides the best alternative for Thermo Fisher in the detection of C. burnetii specific antibodies in rabbits in Australia. Further trials are required to confirm these preliminary results due to the low seroprevalence of Coxiella burnetii observed in the study sera.

Novel multiparameter correlates of Coxiella burnetii infection and vaccination identified by longitudinal deep immune profiling.

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.