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

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

Genetic variation in TLR10 is not associated with chronic Q fever,despite the inhibitory effect of TLR10 on Coxiella burnetii-induced cytokines in vitro

Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10 influences the development of chronic Q fever. HEK293 cells, transfected with TLR2, TLR10 or TLR2/TLR10, and human peripheral blood mononuclear cells (PBMCs) in the presence of anti-TLR10, were stimulated with C. burnetii. In both assays, the absence of TLR10 resulted in increased cytokine responses after C. burnetii stimulation. In addition, the effect of single nucleotide polymorphisms (SNPs) in TLR10 was examined in healthy volunteers whose PBMCs were stimulated with C. burnetii Nine Mile or the Dutch outbreak isolate C. burnetii 3262. Individuals bearing SNPs in TLR10 displayed increased cytokine production upon C. burnetii 3262 stimulation. Furthermore, 139 chronic Q fever patients and 220 controls were genotyped for TLR10 N241H, I775V and I369L. None of these polymorphisms were associated with increased susceptibility to chronic Q fever. In conclusion, TLR10 has an inhibitory effect on in vitro cytokine production by C. burnetii, but the presence of TLR10 polymorphisms does not lead to an increased risk of developing chronic Q fever.     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Efficient Method of Cloning the Obligate Intracellular Bacterium Coxiella burnetii

Coxiella burnetii is an obligate intracellular bacterium that replicates in a large lysosome-like parasitophorous vacuole (PV). Current methods of cloning C. burnetii are laborious and technically demanding. We have developed an alternative cloning method that involves excision of individual C. burnetii-laden PVs from infected cell monolayers by micromanipulation. To demonstrate the cloning utility and efficiency of this procedure, we coinfected Vero cells with isogenic variants of the Nine Mile strain of C. burnetii. Coinhabited PVs harboring Nine Mile phase II (NMII) and Nine Mile phase I (NMI) or Nine Mile crazy (NMC) were demonstrated by immunofluorescence. PVs were then randomly excised from cells coinfected with NMI and NMC by micromanipulation, and PVs harboring both strains were identified by PCR. Fresh Vero cells were subsequently infected with organisms from coinhabited PVs, and the PV excision and PCR screening process was repeated. Without exception, PVs obtained from second-round excisions contained clonal populations of either NMII or NMC, demonstrating that micromanipulation is an efficient and reproducible procedure for obtaining C. burnetii clones.     

Resident Alveolar Macrophages Are Susceptible to and Permissive of Coxiella burnetii Infection

Coxiella burnetii, the causative agent of Q fever, is a zoonotic disease with potentially life-threatening complications in humans. Inhalation of low doses of Coxiella bacteria can result in infection of the host alveolar macrophage (AM). However, it is not known whether a subset of AMs within the heterogeneous population of macrophages in the infected lung is particularly susceptible to infection. We have found that lower doses of both phase I and phase II Nine Mile C. burnetii multiply and are less readily cleared from the lungs of mice compared to higher infectious doses. We have additionally identified AM resident within the lung prior to and shortly following infection, opposed to newly recruited monocytes entering the lung during infection, as being most susceptible to infection. These resident cells remain infected up to twelve days after the onset of infection, serving as a permissive niche for the maintenance of bacterial infection. A subset of infected resident AMs undergo a distinguishing phenotypic change during the progression of infection exhibiting an increase in surface integrin CD11b expression and continued expression of the surface integrin CD11c. The low rate of phase I and II Nine Mile C. burnetii growth in murine lungs may be a direct result of the limited size of the susceptible resident AM cell population.     

Proteomic and Systems Biology Analysis of the Monocyte Response to Coxiella burnetii Infection

Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and C. burnetii is considered a significant bioterror threat. C. burnetii occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of C. burnetii and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.     

Diagnosis of Coxiella burnetii Infection: Comparison of a Whole Blood Interferon-Gamma Production Assay and a Coxiella ELISPOT

Diagnosis of ongoing or past infection with Coxiella burnetii, the causative agent of Q fever, relies heavily on serology: the measurement of C. burnetii-specific antibodies, reflecting the host’s humoral immune response. However, cell-mediated immune responses play an important, probably even more relevant, role in infections caused by the intracellular C. burnetii bacterium. Recent studies have investigated interferon-gamma (IFN-γ) based assays, including a whole-blood IFN-γ production assay and a Coxiella enzyme-linked immunospot (Coxiella ELISPOT), as potential diagnostic tools for Q fever diagnosis. Both are in-house developed assays using stimulating antigens of different origin. The main objective of this study was to compare the test performance of the IFN-γ production assay and the Coxiella ELISPOT for detecting a cellular immune response to C. burnetii in Q fever patients, and to assess the correlation between both assays. To that end, both tests were performed in a well-defined patient group of chronic Q fever patients (n = 16) and a group of healthy seronegative individuals (n = 17). Among patients, both the Coxiella ELISPOT and the IFN-γ production assay detected positive response in 14/16. Among controls, none were positive in the Coxiella ELISPOT, whereas the IFN-γ production assay detected positive results in 1/17 and 3/17, when using Henzerling and Nine Mile as stimulating antigens, respectively. These results suggest the Coxiella ELISPOT has a somewhat higher specificity than the IFN-γ production assay when Nine Mile is used as antigen stimulus. The assays showed moderate correlation: the Spearman correlation coefficient r ranged between 0.37–0.60, depending on the antigens used. Further investigation of the diagnostic potential for C. burnetii infection of both assays is warranted.     

A Probably Minor Role for Land-Applied Goat Manure in the Transmission of Coxiella burnetii to Humans in the 2007–2010 Dutch Q Fever Outbreak

In 2007, Q fever started to become a major public health problem in the Netherlands, with small ruminants as most probable source. In order to reduce environmental contamination, control measures for manure were implemented because of the assumption that manure was highly contaminated with Coxiella burnetii. The aims of this study were 1) to clarify the role of C. burnetii contaminated manure from dairy goat farms in the transmission of C. burnetii to humans, 2) to assess the impact of manure storage on temperature profiles in dunghills, and 3) to calculate the decimal reduction time of the Nine Mile RSA 493 reference strain of C. burnetii under experimental conditions in different matrices. For these purposes, records on distribution of manure from case and control herds were mapped and a potential relation to incidences of human Q fever was investigated. Additionally, temperatures in two dunghills were measured and related to heat resistance of C. burnetii. Results of negative binomial regression showed no significant association between the incidence of human Q fever cases and the source of manure. Temperature measurements in the core and shell of dunghills on two farms were above 40°C for at least ten consecutive days which would result in a strong reduction of C. burnetii over time. Our findings indicate that there is no relationship between incidence of human Q fever and land applied manure from dairy goat farms with an abortion wave caused by C. burnetii. Temperature measurements in dunghills on two farms with C. burnetii shedding dairy goat herds further support the very limited role of goat manure as a transmission route during the Dutch human Q fever outbreak. It is very likely that the composting process within a dunghill will result in a clear reduction in the number of viable C. burnetii.     

Genotypic and phenotypic characterization of two Swedish isolates and two prototypic strains of Coxiella burnetii

Two Swedish isolates of Coxiella burnetii and the two prototype strains of the species, Nine Mile and Priscilla, were characterized with regard to their multiplication and cytopathic effect on BGM cells and by PCR-based amplification of repetitive element DNA and the C. burnetii -specific plasmids QpH1 and QpRS. Moreover, 1330 bp of each 16S rRNA gene were sequence-determined. All four strains multiplied at virtually the same rate and displayed the same type of vacuoles in the BGM cells. Genetic homogeneity was observed inasmuch as the 16S rDNA sequences were identical and the strains showed identical PCR amplification patterns using primers specific to enterobacterial repetitive intragenic consensus DNA sequences. The two Swedish strains and the Priscilla strain also showed identical patterns after PCR amplification of repetitive extragenic palindromic DNA sequences, whereas the Nine Mile strain demonstrated a similar, but not identical pattern. Thus, the investigated strains demonstrated very similar phenotypic and genotypic characteristics. This finding is discussed in view of the very rare occurrence of domestic Q fever in Sweden.     

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.     

Isolation of Coxiella burnetii from Dairy Cattle and Ticks,and Some Characteristics of the Isolates in Japan

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty-nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.     

IS<Emphasis Type="Italic">1111</Emphasis> insertion sequences of <Emphasis Type="Italic">Coxiella burnetii</Emphasis>: characterization and use for repetitive element PCR-based differentiation of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> isolates

Background  

Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.     

Persistent High IgG Phase I Antibody Levels against Coxiella burnetii among Veterinarians Compared to Patients Previously Diagnosed with Acute Q Fever after Three Years of Follow-Up

Background

Little is known about the development of chronic Q fever in occupational risk groups. The aim of this study was to perform long-term follow-up of Coxiella burnetii seropositive veterinarians and investigate the course of IgG phase I and phase II antibodies against C. burnetii antigens and to compare this course with that in patients previously diagnosed with acute Q fever.

Methods

Veterinarians with IgG phase I ≥1:256 (immunofluorescence assay) that participated in a previous seroprevalence study were asked to provide a second blood sample three years later. IgG antibody profiles were compared to a group of acute Q fever patients who had IgG phase I ≥1:256 twelve months after diagnosis.

Results

IgG phase I was detected in all veterinarians (n = 76) and in 85% of Q fever patients (n = 98) after three years (p<0.001). IgG phase I ≥1:1,024, indicating possible chronic Q fever, was found in 36% of veterinarians and 12% of patients (OR 3.95, 95% CI: 1.84–8.49).

Conclusions

IgG phase I persists among veterinarians presumably because of continuous exposure to C. burnetii during their work. Serological and clinical follow-up of occupationally exposed risk groups should be considered.     

Genome‐wide profiling of humoral immune response to Coxiella burnetii infection by protein microarray

Comprehensive evaluation of the humoral immune response to Coxiella burnetii may identify highly needed diagnostic antigens and potential subunit vaccine candidates. Here we report the construction of a protein microarray containing 1901 C. burnetii ORFs (84% of the entire proteome). This array was probed with Q‐fever patient sera and naïve controls in order to discover C. burnetii‐specific seroreactive antigens. Among the 21 seroreactive antigens identified, 13 were significantly more reactive in Q‐fever cases than naïve controls. The remaining eight antigens were cross‐reactive in both C. burnetii infected and naïve patient sera. An additional 64 antigens displayed variable seroreactivity in Q‐fever patients, and underscore the diversity of the humoral immune response to C. burnetii. Nine of the differentially reactive antigens were validated on an alternative immunostrip platform, demonstrating proof‐of‐concept development of a consistent, safe, and inexpensive diagnostic assay alternative. Furthermore, we report here the identification of several new diagnostic antigens and potential subunit vaccine candidates for the highly infectious category B alphaproteobacteria, C. burnetii.     

Microevolution of the Chromosomal Region of Acute Disease Antigen A (adaA) in the Query (Q) Fever Agent Coxiella burnetii

The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.     

Antigenic Characteristics of Polypeptides of Coxiella burnetii Isolates

Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti-C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.     

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

Background  

Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA).     

Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages

The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome‐like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii–host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non‐fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro‐survival kinases and short‐termphosphorylation of stress‐related p38. Avirulent organisms also triggered a robust, early pro‐inflammatory response characterized by increased secretion of TNF‐α and IL‐6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro‐ and mature IL‐1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN‐γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease‐relevant platform for defining novel innate immune responses to C. burnetii.     

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

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