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Q fever epidemiology and pathogenesis
Microbes and Infection, 2, 2000, 417−424 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
Review
Q fever epidemiology and pathogenesis Lena Norlander Defence Research Establishment, Division of NBC Defence, SE-901 82 Umeå, Sweden
ABSTRACT – The lungs are a port of entry and primary infectious focus of Coxiella burnetii, the obligate intracellular contagium of the worldwide zoonosis Q fever. The infectious process and immune response are characterised by studies in cell culture and animal systems. Following endocytosis, replication exclusively occurs in the phagolysosome. Several potential virulence factors are described. © 2000 Éditions scientifiques et médicales Elsevier SAS Q fever / Coxiella burnetii / epidemiology / pathogenesis / host response
1. Introduction
2. Epidemiology
Q fever is a worldwide zoonosis caused by Coxiella burnetii, an obligatory intracellular organism which is a member of the family Rickettsiaceae [1]. Recently, the use of molecular biology techniques has shown that C. burnetii is phylogenetically distant from the other members of this family [2]. C. burnetii belongs to the gamma subdivision of the Proteobacteria. C. burnetii is widely distributed in nature and responsible for infection in various mammals, including humans, and in birds, reptiles and fish [3, 4]. Humans beings are, however, the only host known to develop illness as a result of infection. Infection in animals is mainly subclinical but has been associated with late abortions, stillbirth, delivery of weak offspring and also infertility [5]. The microorganism is maintained in nature by two cycles that are essentially independent but occasionally overlapping. The basic cycle involves many species of wildlife and their ectoparasites, and the second cycle domestic animals [5]. Ticks are considered to be the natural primary reservoirs of C. burnetii responsible for the spread of the infection in wild animals and for transmission of C. burnetii from wild to domestic animals. Among domestic animals, cattle, sheep and goats are considered to be the main reservoirs of the agent responsible for infection of humans. Infected female animals shed large numbers of C. burnetii through their placenta and birth fluid, colostrum and milk into the environment. The excretions are potential sources of infection in animals and humans via inhalation of infectious aerosol of airborne dust. Estimates of infectivity for humans range from 1 to 10 bacteria. Since C. burnetii is very resistant and little affected by extreme environmental conditions, it can form a highly infectious dust and it remains viable over long period of time [5].
In 1955 Q fever was known to occur in 51 countries on five continents, and in the last decade the disease had been reported in yet another 8 countries [6]. Furthermore, seroepidemiological surveys have indicated the presence of Q fever in several other countries. New Zealand remains probably the only country in the world where Q fever is absent [7]. There is a remarkable diversity in the epidemiology of Q fever in different geographical regions. In most European countries Q fever attracts relatively low attention because of the low incidence of disease [5, 6]. The outbreaks during the last decade have usually been of limited size and in many cases the source of the Q fever infection is not identified. The largest outbreak of acute Q fever in the UK occurred in 1989. The most likely route of infection for the 147 diagnosed cases was assumed to be windborne spread from farmland to an urban area. Sheep and cattle in the UK are endemically infected with C. burnetii [8]. In northern Italy an increased number of Q fever cases was correlated to indirect exposure to sheep, i.e., by aerosol exposure to the infectious agent in the neighbourhood of farms [9]. A study of a German Q fever outbreak also suggested airborne transmission of C. burnetii from a large sheep farm after lambing season [10]. The results demonstrated that many residents were infected but had minimal or no symptoms. France has a high prevalence of acute Q fever, which is assumed to reflect a high national awareness of the disease. Chronic Q fever is also more frequently reported from France than other countries [11]. In contrast, in the former Czechoslovakia and the present Slovakia, several hundred cases of acute Q fever have been reported during the last two decades, but no case of chronic Q fever has been confirmed. The correlation between infected domes-
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tic animals and Q fever has been established, i.e., the source of a Q fever outbreak in Slovakia affecting more than a hundred persons was shown to be imported goats [12]. In Bulgaria, goats have been concluded to be the main reservoir for C. burnetii in recent years [13]. Hundreds of serologically confirmed cases of acute Q fever have occurred and chronic forms of the disease have also been observed. Studies of the epizootiological and epidemiological situation of Q fever in Poland indicated C. burnetii to be common among sheep and cattle, but few human cases have been reported [14]. Extremely few cases of domestic Q fever have been diagnosed in Sweden, but the contagium has been isolated from sheep placentas and from mouldy hay ( [15], Macellaro, unpublished data). A recent survey of Q fever showed that sheep farmers and veterinarians were risk groups for exposure to C. burnetii [16]. Similar observations, i.e., a strong correlation between domestic animals and limited epidemics/sporadic cases of Q fever, are reported also from other continents than Europe. Serosurveys on the occurrence of C. burnetii in central and eastern Canada, showed a high prevalence of antibodies to the contagious agent in blood donors [17]. In one of these regions no case of Q fever had never been reported, while sporadic cases appeared in the other region. Russia has Q fever with foci of different intensity in large cattle and sheep breeding complexes and farms and also in poultry factories [18]. But there is also a high prevalence of antibodies to C. burnetii among individuals that have no professional contact with animal or animal products, which indicates a change in the epidemiological patterns of Q fever [19]. The infection is widespread in Japan, where domestic animals have been suggested to be important reservoirs of C. burnetii [20].
3. C. burnetii infection in humans Human infection with C. burnetii can be either subclinical, acute or chronic [21]. Asymptomatic Q fever infection is common, since the number of serologically positive persons is much higher than the incidence rate of the disease [5]. The incubation time for acute Q fever ranges from two to four weeks. In acute Q fever the symptoms resemble those of influenza (fever, headache and myalgia), and the disease may become complicated with pneumonia and/or granulomatous hepatitis. Cases of febrile reruptions, myocarditis, pericarditis and meningoencephalitis have been reported [21]. C. burnetii has been isolated from placenta and breast milk from mothers who have had an acute Q fever infection a few years before delivery. This indicates that resting C. burnetii undergoes reactivation during pregnancy. Some of the mothers gave birth to healthy children, while others suffered miscarriages and delivery of abnormal children [21]. Chronic Q fever, which is most often characterized by endocarditis, may develop years after an acute infection. In contrast to acute Q fever, the mortality rate for chronic Q fever has been reported to be high [21]. The development of acute or chronic Q fever infection is assumed to depend on the patient’s condition and 418
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immune status rather than on specific ’acute’ or ’chronic’ C. burnetii strains [22, 23]. If acute Q fever is diagnosed, antibiotic treatment is recommended, but the disease usually also resolves without treatment. Tetracycline compounds, especially doxycycline and also quinolone compounds are currently used to treat Q fever. In chronic Q fever, treatment with a combination of several antibiotics lasting from one to several years is necessary [21, 24]. Experimental infection of animals indicates that the route of infection determines the predominant manifestations of the acute form of the disease [25, 26]. Intraperitoneal inoculation of C. burnetii in guinea pigs led to pathologic changes mainly in the liver, whereas intranasal infection caused pathologic changes mainly in the lungs. The size of the inoculum influenced clinical expression in acute Q fever [26]. A correlation between route of infection and the predominant symptoms could explain the variation in manifestations of acute Q fever in different geographical areas. In southeast Canada, Switzerland and northern Spain the main manifestation of acute Q fever is pneumonia, assumed to be caused by inhalation of contaminated aerosols. In contrast, in France or southern Spain it is granulomatous hepatitis which has been speculated is due to caused by ingestion of contaminated raw milk [26].
4. The contagious agent of Q fever The aetiological agent of Q fever, C. burnetii, is an obligate intracellular bacterium and during the infectious process, it is taken up by host cells into phagosomes, which then fuse with primary lysosomes to form phagolysosomes. C. burnetii is unique among the pathogenic bacteria because it has evolved biochemical strategies of growth in the hostile acidic phagolysomes of host cells. Actually, the acidic content of the phagolysosome is a requisite for its growth and multiplication [27]. C. burnetii has a cell wall similar to that of Gramnegative bacteria and it is characterised by a phase transition [1, 28]. C. burnetii phase I, which is isolated from infected humans or animals, has a smooth hydrophilic lipopolysaccharide (LPS) surface structure. Upon cultivation in tissue cell cultures or in embryonated hen’s eggs, this smooth type of LPS is lost and the surface of the phase II variant contains truncated rough-type LPS molecules with a hydrophobic surface. The phase transition is accompanied by a loss of virulence [29]. A mutant with an LPS of intermediate complexity has been described. The LPS of C. burnetii has an endotoxic activity which is 100 to 1 000 times less than the LPS of Enterobacteria [30]. The extreme stability of C. burnetii has been associated with the small dense cell forms and spore-like forms observed in electron microscopy studies [31]. C. burnetii has a developmental cycle which consists of both vegetative growth by binary fission and unequal cell division leading to spore-like formation. This cycle results in the formation of a heterogeneous mixture of pleomorphic cell types with morphologically distinguishable cell variants. The compact small cell variants (SCV) (0.2–0.5 µm) are Microbes and Infection 2000, 417-424
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Figure 1. Electron micrograph of a Buffalo green monkey cell heavily infected by C. burnetii.
metabolically dormant and they are assumed to survive extracellularly for long periods. In contrast, the large cell variants (LCV) (≥1.0 µm) have a high metabolic activity and have lost the resistant properties. Both small and large cell variants are infectious in in vitro and in vivo models. Several proteins that are differentially synthesized by the two variants have been characterized and these are described in a recent review [32]. The spore-like forms have been observed in the large cell variants and the formation is thought to occur by a process that resembles asymmetric cell division [31]. Spore-like formation has also been detected in C. burnetii-infected cardiac valves [33]. Moreover, a partial sequence of a sporulation gene in C. burnetii has recently been published [34]. Spore particles have, however, not yet been prepared in pure form.
5. Entry into and proliferation in host cells C. burnetii infects a great number of cell types including monocytes, macrophages and a variety of transformed cells [1]. The ability to efficiently invade and subsequently grow within eukaryotic cells is a key virulence factor for organisms, which require an intracellular environment for their multiplication. C. burnetii could be expected to utilize several cell processes for uptake – endocytosis – in a similar way to that of the obligate intracellular species of Chlamydiaecae [35]. Several publications have reported that C. burnetii reaches its internal niche by phagocytosis and it enters passively [1, 36]. The phase II bacteria are more readily internalised by the host cells than are the phase I variants. Opsonisation by specific Ig enhances phagocytosis of both phase I and II C. burnetii. Moreover, based on recent observations, additional variants of endocytosis have been suggested to be used in C. burnetii internalisation. Meconi et al. [37] showed that virulent, but not avirulent, C. burnetii stimulated morphological Microbes and Infection 2000, 417-424
changes in monocytes similar to those observed in macropinocytosis of Salmonella typhimurium. Upon contact between C. burnetii and the host cell membrane, protrusions and polarized projections are induced over an extensive region of the cell surface. In addition, studies on the efficient internalisation of the phase II variants into macrophages after treatment by inhibitors of endocytosis indicate that pinocytic pathways are involved [38]. The simulataneously use of various endocytic variants for its internalisation in a wide spectrum of host cells offers an advantage for C. burnetii in establishing infection. C. burnetii is a slowly growing bacterium with a generation time of several hours. Studies of C. burnetii growth in tissue cell cultures show proliferation of bacteria in vacuoles which may fuse and form a huge single vacuole. This vacuole can occupy most of the enlarged cell’s volume and the nucleus and cytoplasm are displaced to the outermost part of the infected cell. In spite of the bacterial burden, such a cell seems to grow almost normally [1] (figure 1).
6. Virulence factors and their genes Though C. burnetii has been recognised worldwide as an important pathogen, there is limited knowledge of its virulence genes. The functions and activities of proteins on the C. burnetii cell surface and proteins exported to the exterior growth environment are likely to be important for understanding aspects of its intracellular survival and growth. C. burnetii protein synthesis can be studied in a cell-free acidic system, where nucleic acid and protein synthesis as well as a minor increase in cell size are demonstrated [39]. Growth, however, by the definition doubling of both mass and number of cells, does not occur in this in vitro system. Incubation of C. burnetii in the acidic in vitro system rapidly triggers the synthesis of a number of proteins [39]. This in vitro activation of protein synthesis is assumed to 419
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reflect the initial production of proteins in vivo and thus, is a tool to study proteins of significance for the early phase of infection. Among these proteins are heat shock protein 60 and 70 homologues, Hsp62 and Hsp71 [40, 41]. The Hsp62 gene htpB is colocalized with htpA. The genes htpA and htpB correspond to the groES and groEL of Escherichia coli [40]. The Hsp70 homologue in C. burnetii is the hsp71 gene product, a protein with high homology to the E. coli DnaK protein [41]. Both htpB and hsp71 are under the regulation of a promoter with a strong sequence homology to the E. coli consensus heat shock promoter. Hsp71 has been shown to be cell wallassociated in addition to the cytosolic localization expected for chaperones [41]. It is not known if the dual localization in C. burnetii reflects dual roles of Hsp71. Additional functions of chaperons of the Hsp60 and Hsp70 families of other pathogens have been suggested, i.e., invasion in cell culture models. Several prokaryotic heat shock proteins have also been shown to be important immunogens and to stimulate both T cells and B cells [42]. Both the 62- and 71-kDa Hsp of C. burnetii are reported to be immunodominant [40, 41]. A third heat shock protein gene, dnaJ, has also been described in C. burnetii [43]. Another C. burnetii protein, the mucZ gene product, shares C-terminal sequence homology with the J domain of the DnaJ protein, and it was demonstrated to induce capsule synthesis in E. coli [44]. Potential interactions are suggested between MucZ and the DnaK-chaperon machinery and a requirement for DnaK is indicated. The observed surface localization of Hsp71 may indicate a role of the protein delivery of the mucZ gene product to the surface. A possible virulence property of C. burnetii is the demonstrated active secretion of proteins. In vitro studies suggest that specific secretion of selected proteins from the bacteria occurs a few hours after exposure to an acidic environment [45]. This might reflect a mechanism for efficient delivery of selected bacterial proteins to the interior of the host cell, thus opening a possibility for the bacterium to ’communicate’ with its host. In Q fever, macrophages play a central role, since they are host cells and involved in the dissemination of C. burnetii. By looking for sequences of potential virulence compounds of other pathogens with similar infection niches, a number of C. burnetii genes have been isolated. One example is the macrophage infectivity potentiator gene, cbmip [46]. Mip has been demonstrated in several intracellular pathogens such as Legionella pneumophila and Chlamydia trachomatis, where it has been shown to have a role as a virulence factor. The enzymatic activity of Mip proteins (PPIase) appears to be related to the ability of intracellular organisms to initiate infection. Further characterization of cbmip revealed a mechanism to optimise the utilization of the limited genome capacity of C. burnetii (about one third of the E. coli genome). Mo et al. identified two additional proteins, which were synthesized from the same open reading frame, but starting at different internal translation start codons [47]. All three proteins exhibit similar PPIase activities. The product of C. burnetii qrsA (Q fever agent regulatory sensor-like gene) has also been identified as a homo420
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logue to a virulence gene of pathogens [48]. QrsA is a PhoQphoR homologue, which is involved in regulation of acid phosphatase gene expression in Salmonella. C. burnetii has been shown to produce acid phosphatase activity which could account for its observed capacity to inhibit the oxidative burst in neutrophils [49]. Moreover, C. burnetii possesses superoxide dismutase and catalase, which confer protection from the microbicidal effects of reactive oxygen intermediates of the host cells [50]. The factors involved in the bacterial differentiation confer a flexibility of great value for C. burnetii. A number of proteins, which are differentially expressed in SCV and LCV, have been described [32, 51]. ScvA and Hq1 are SCV-specific proteins that are localized to the cytoplasm. These proteins are assumed to have structural roles in the formation of the condensed SCV nucleoid. They are degraded during development of the SCV to the LCV. The outer membrane proteins OMP34 and P1 are differentially synthesized in the two cell variants. At least two other differentially expressed proteins have been identified, i.e., homologues to the elongation factors (EF) EF-Tu and EF-Ts.
7. Genome and plasmids of C. burnetii Recently, a physical and genetic map was constructed of the 2 103-kb C. burnetii linear chromosome [52]. Open reading frames with homology to database entries and genes were located on the physical map by southern hybridization. The slow growth of C. burnetii is assumed to be a reflection of the single rRNA operon copy, since the presence of a low copy number of the rRNA operon is a trend among slow-growing bacteria, including other obligate intracellular bacteria [53]. Several authors have reported the existence of cryptic plasmids in C. burnetii. Initial studies identified the first C. burnetii plasmid, the 36-kb QpH1, from the American isolate Nine Mile [54]. The entire nucleotide sequence of this plasmid has been deduced [55]. Screening of a large number of isolates from different geographic sources has also shown the existence of other plasmids in C. burnetii. The larger QpRS (38–39 kb) and QpDG (51 kb) as well as the slightly smaller QpDV plasmids and QpH1 have 25 to 39-kb regions, which share a large amount of homology [55, 56]. These sequences are also present in the chromosome of a plasmidless C. burnetii isolate [57]. Comparison of the entire QpH1 plasmid sequence of phase I and II, and plasmid sequences integrated into the chromosome of a plasmidless strain phase I and II, revealed no evidence for specific genes or sequences involved in phase variation [55, 57]. Furthermore, the general view today is that there is no correlation between plasmid type of the infectious agent and acute or chronic form of the disease [22, 23]. Moreover, genetic characterisation of isolates from patients with acute and chronic Q fever demonstrated a considerable heterogeneity between the isolates, and they could not be grouped into acute and chronic strains. Restriction fragment length polymorphism was used to differentiate 80 C. burnetii isolates from various geographical origins. This revealed 20 different restriction Microbes and Infection 2000, 417-424
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groups and evolutionary relationships among groups, which corresponded to the geographical origin of the isolates [58].
8. Infection models of Q fever The predominant form of the disease is the acute form of Q fever. During an acute Q fever infection any organ may be involved, but the lungs and liver are most often affected. The lungs not only serve as a portal of entry of the pathogen into the human body, but also an area of primary infectious focus. In the lung, the principal defensive mechanism is the clearance of the particulate microorganisms by either the mucociliary process in conducting airways or the transport of the microorganisms from the lung to the regional tracheobronchial lymph nodes. The mobile lung macrophages may participate in the systemic spread of C. burnetii by retaining them in their intracellular acidic vesicles. The most common animal model systems for studies of Q fever are guinea pigs and mice. The mouse model of Q fever is attractive to use for studies of airborne infection of C. burnetii since there is a considerable similarity among mammalian species in overall lung composition and in the distribution and features of cells of the respiratory system. There are several publications concerning experimental intranasal or aerosol infection of mice with C. burnetii [59-61]. Symptoms of disease are described as lethargy, ruffled fur and, in some sensitive mice strains, even death. Pneumonia and hepatitis may also be observed. The infectious process in lungs of sensitive mice has been described as either interstitial or intra-alveolar inflammation with the macrophages prevailing in the excudate. There is proliferation of bronchiolar epithelium and desquamation of cells. The infection is self-limiting with a complete resolution and also associated with the influx of T cells in the pneumonic foci. In mouse liver focal mononuclear cell aggregates are observed. Gross lesions consisting of enlargement of spleen have been reported one to two weeks postinfection. In guinea pigs infected by C. burnetii the gross pathology of liver has a characteristic pattern such as pyrexia, development of hepatomegaly and fatty infiltration [62]. Studies on the pathobiochemical events and their regulation during Q fever in guinea pigs showed specific effects on hepatic metabolic processes. Stimulated hepatic transcription and translation of certain RNA and protein species attend the development of the disease, as do phosphorylation and dephosphorylation of central RNA and protein species.
9. Innate and acquired immunity Immunity to intracellular microbial pathogens is assumed to be essentially due to cell-mediated defence development [63]. The successful elimination of such pathogens depends on the activation of the appropriate blend of T cells and resistance correlates with a Th1dominated immune response. There is a close interaction Microbes and Infection 2000, 417-424
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between the innate and acquired immune response, i.e., the divergence into Th1 and Th2 cells is regulated by the innate immune system. Macrophages have important immunoregulatory functions in development of protective immunity. Proinflammatory cytokines such as interleukin 1 (IL-1), tumour necrosis factor (TNF) and interleukin 12 (IL-12), which are secreted by infected macrophages, are capable of modulating the cellular immune response. Gene-knockout studies of cytokines and their receptors have verified the essential role of interferon γ (IFN-γ) and TNF in defence against intracellular pathogens [63]. A number of observations verify the requirement of cell-mediated response for clearance of C. burnetii infection also. It has been shown that infection of athymic mice (lacking T cells) by C. burnetii produces a progressive nonlethal disease, while normal mice cleared the infection in a few weeks [59]. Moreover, chronic Q fever is developed by humans with defective immunological status and T-cell-mediated immunity is depressed in Q fever endocarditis [64]. The role of antibodies in Q fever is controversial, but most investigators support the view that antibodies are not required for control of C. burnetii infection. Antibodies, however, may contribute to the elimination of C. burnetii, i.e., in the immune host, professional phagocytes are influenced by antibodies. Moreover, C. burnetii phase II organisms are more exposed for binding of opsonising antibodies than are phase I bacteria, whose LPS masks surface antigens from the immune system [65]. The preferential killing of avirulent phase II bacteria is also suggested to be due to binding of complement C3b [66]. Phase I organisms resist complement-mediated serum killing and this appears to be correlated with the LPS. In vitro and in vivo studies demonstrate that IFN-γ and TNF play important roles in the elimination of C. burnetii [67-69]. Growth of C. burnetii in a monocytic cell line was inhibited by IFN-γ and this cytokine also induced apoptosis of infected monocytes [67]. IFN-γ-mediated killing of C. burnetii and death of infected monocytes were both dependent on TNF. Macrophages infected in vitro by virulent C. burnetii had an early and strong induction of TNF-α [70]. The triggering of TNF-α was equivalent in cells infected by C. burnetii phase I and phase II. C. burnetii in macrophages would have a survival advantage by downregulating the IFN-γ production, which otherwise would activate the host cell. In vitro studies have shown that the IFN-γ production is inhibited when C. burnetii phase I LPS interacts with monocytes and macrophages [71]. Modification of phase I LPS sugar chains to form an artificial phase II LPS sharply increases IFN-γ when exposed to the test system, even though the protein composition remains the same [71]. These findings are in agreement with the observation of experimental infection caused by C. burnetii phase I and phase II, respectively. The phase II bacteria are more readily eliminated and this might in part be due to a less efficient restriction of the IFN response. The phase I-infected macrophages also produced IL-1α. In contrast, no similar IL-1α induction was found in macrophages infected by avirulent phase II bacteria. The 421
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differential induction of IL-1α by the two variants could not be correlated to LPS purified from phase I or phase II C. burnetii, respectively [70]. The early IL-1α induction in macrophages infected by C. burnetii phase I, but not phase II, indicates a specific role of the cytokine in the infectious process. When the levels of circulating inflammatory cytokines and their antagonists were determined in leucocytes of Q fever patients, the level of TNF was found to be markedly increased [69]. The high TNF level was most pronounced in blood from patients with chronic Q fever (endocarditis), while the levels in acute Q fever cases were lower. Moreover, the increased TNF levels are assumed to result in the observed upregulation of TNF receptor type II. There was also increased levels of IL-1 receptor antagonist, which is known to block the activity of IL-1 [69]. This may contribute to a decreased resistance to C. burnetii infection.
10. Conclusion Q fever attracts relatively low attention in most countries because of the moderate disease incidence rate and the limited size of the outbreaks. It is often impossible to identify the source of infection, but domestic animals are considered to be the main reservoirs of C. burnetii responsible for infection of humans. Characterisation of isolates from various geographical sources revealed evolutionary relationships among groups that correspond to the geographical origin of the isolate. The development of human infection, which is either acute or chronic, is correlated to the patient’s condition and immune status rather than to specific ’acute’ or ’chronic’ C. burnetii strains. In acute Q fever, which is the most common manifestation of the disease, the symptoms resemble those of influenza. The route of infection, i.e., by aerosol or oral, determine the predominant symptoms. Chronic Q fever is most often characterized by endocarditis and it may develop years after an acute infection. Acute Q fever usually resolves without treatment, while chronic infection requires prolonged treatment with a combination of several antibiotics. In vitro and in vivo studies demonstrate that IFN-γ and TNF play important roles in the host defence processes leading to the elimination of C. burnetii. Recent observations also indicate the involvement of IL-1. Cell-mediated immune response is required for clearance of a C. burnetii infection and T cell-mediated immunity is shown to be depressed in Q fever endocarditis. Moreover, data on circulating inflammatory cytokines and receptor antagonists suggest a shift of cytokine balance towards cytokine antagonists in acute Q fever. This may contribute to a decreased resistance to C. burnetii infection. C. burnetii is characterised by a phase transition phenomenon involving LPS. The phase transition is accompanied by a loss of virulence. So far, the LPS is the only component verified to differ between the virulent phase I and avirulent phase II of C. burnetii. The bacterium has an extreme stability, which has been explained by the different morphological forms of its developmental cycle, i.e., the non-metabolic SCV and the endospore-like forms. The 422
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metabolically active LCV has lost the resistant properties. Both cell variants are infectious in vitro and in vivo. Several proteins that are differentially synthesized by the two cell variants have been characterized. It is to be assumed that these proteins are important contributions to the ability of C. burnetii to act as an efficient contagious agent. C. burnetii has an unique adaptation to the harsh acidic milieu of the phagolysosomes, to which it is delivered after endocytosis. Recent reports indicate the use of various endocytic pathways for efficient internalisation, which should be a prerequisite for a successful obligate intracellular bacterium. Physical and genetic maps have been constructed for the 2 103-kb C. burnetii linear chromosome as well as for the crypic plasmid variants, which share large regions of homology. There is no evidence for specific genes or sequences involved in the phase transition or in disease manifestation. The absolute requirement for low pH has been utilised for in vitro studies of protein induction and these studies have demonstrated the production of several Hsp being essential proteins and potential virulence factors, i.e., homologues to E. coli GroEL, GroES, DnaK and DnaJ. Screening of gene libraries for other potential virulence factors, such as factors required for intracellular survival, has successfully demonstrated a number of such proteins. Examples are three slightly different macrophage potentiator factors and a regulatory sensor-like protein which is suggested to have a role in regulation of acid phosphatase. The acid phosphatase may account for inhibition of the intracellular bactericidal oxidative burst. Moreover, C. burnetii possesses superoxide dismutase and catalase, which confer protection from the microbiocidal effects of reactive oxygen intermediates of the host cells. Taken together, however, there are so far few ’virulence factors’ described for C. burnetii. This is assumed to be due to the problems concerning studies on intracellular organisms. For the future, genome sequencing and the recently described genetic system for C. burnetii are promising tools for studies to increase the knowledge of the pathogenic nature of this organism.
Acknowledgments I am grateful to Karin Hjalmarsson and Anna Macellaro for helpful discussions and critical reading of the manuscript and to Lenore Johansson for the electron microscopic photo.
References [1] Baca O.G., Paretsky D., Q fever and Coxiella burnetii: a model for host-parasite interactions, Microbiol. Rev. 46 (1983) 127–149. [2] Stein A., Saunders N.A., Taylor A.G., Raoult D., Phylogenic homogenicity of Coxiella strains as determined by 16S ribosomal RNA sequencing. FEMS Microbiol. Lett. 113 (1993) 339–244. Microbes and Infection 2000, 417-424
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[3] Sawyer L.A., Fishbein D.B., McDade J.E., Q fever – current concepts, Rev. Infect. Dis. 9 (1987) 935–946. [4] Rehácek J., Tarasevich I.V., IV Q fever, in: Gresiková M. (Ed.), Acari-borne rickettsiae and rickettsioses in Eurasia, VEDA, Bratislava, 1988, pp. 203–243. [5] Aitken I.D., Bögel K., Cracea E., Edlinger E., Houwers D., Krauss H., Rády M., Rehácek J., Schiefer H.G., Schmeer N., Tarasevich I.V., Tringali G., Q fever in Europe: Current aspects of aetiology, epidemiology, human infection, diagnosis and therapy, Infection 15 (1987) 323–327. [6] Kazár J., Q fever – current concept, in: Raoult D., Brouqui P. (Eds.), Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium, Elsevier, Amsterdam, 1999, pp. 304–319. [7] Hilbink F., Penrose M., Kovácová E., Kazár J., Q fever is absent from New Zealand, Int. J. Epidemiol. 22 (1993) 945–949. [8] Smith D.L., Ayres J.G., Blair I., Burge P.S., Carpenter M.J., Caul E.O., Couplands B., Desselberger U., Evans M., Farrell I.D., Hawker J.I., Smith E.G., Wood M.J., A large Q fever outbreak in the West Midlands: clinical aspects, Resp. Med. 87 (1993) 509–516. [9] Selvaggi T.M., Rezza G., Scagnelli M., Rigoli R. et al., Investigation of a Q fever outbreak in Northern Italy, Eur. J. Epidemiol. 12 (1996) 403–408. [10] Lyytikäinen O., Ziese T., Schwartländer B., Matzdorff P., Kuhnhen C., Jäger C., Petersen L., An outbreak of sheepassociated Q fever in a rural community in Germany, Eur. J. Epidemiol. 14 (1998) 193–199. [11] Raoult D., Q fever: still a query after all these years, J. Med. Microbiol. 44 (1996) 77–78. [12] Kovácová, E., Kazár J., Simková A., Clinical and serological analysis of a Q fever outbreak in western Slovakia with four-year follow-up, Eur. J. Clin. Microbiol. Dis. 17 (1998) 867–869. [13] Serbezov V., Kazár J., Novkirishki V., Gatcheva N., Kovácová E., Voynova V., Q fever in Bulgaria and Slovakia, Emerging Infect. Dis. 5 (1999) 388–394. [14] Anusz Z., Ciecierski H., Knap J., Piesiak Z., Platt-Samoraj A., Siemionek J., Szweda W., Szymamski M., Lecki J., Kowalska E., Q fever in Poland in the year 1952–1995, in: Kazár J., Toman R. (Eds.), Rickettsia and Rickettsial Diseases, Slovak Acad. Sci., Veda Bratislava, Slovakia, 1996, pp. 528–534. [15] Åkesson Å., Krauss H., Thiele H.D., Macellaro A., Schwan, O., Norlander, L., Isolation of Coxiella burnetii in Sweden, Scand. J. Infect. Dis. 23 (1991) 273–274. [16] Macellaro A., Åkesson Å., Norlander L., A survey of Q fever in Sweden, Eur. J. Epidemiol. 9 (1993) 213–216. [17] Marrie T.J., Seroepidemiology of Q fever in New Brunswick and Manitoba, Can. J. Microbiol. 34 (1988) 1043–1045. [18] Rudakov N.V., Ecologico-epidemiological observations of Q fever in Russia, in: Kazár J., Toman R. (Eds.), Rickettsia and Rickettsial Diseases, Slovak Acad. Sci., Veda Bratislava, Slovakia, 1996, pp. 524–527. [19] Rybakova N., Tokarevich N., Chaika N., Q fever monitoring in Vologda province in northern Russia, in: Raoult D., Brouqui P. (Eds.), Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium, Elsevier, Amsterdam, 1999, pp. 351–355. Microbes and Infection 2000, 417-424
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[20] Hirai K., To H., Advances in the understanding of Coxiella burnetii infection in Japan, J. Vet. Med. Sci. 60 (1998) 781–790. [21] Sawyer L.A., Fishbein D.B., McDade J.E., Q fever: current concepts, Rev. Infect. Dis. 9 (1987) 935–946. [22] Yu X., Raoult D., Serotyping Coxiella burnetii isolates from acute and chronic patients by using monoclonal antibodies, FEMS Microbiol. Lett. 117 (1994) 15–19. [23] Thiele D., Willems H., Is plasmid based differentiation of Coxiella burnetii in acute and chronic isolates still valid?, Eur. J. Epidemiol. 10 (1994) 427–434. [24] Raoult D., Treatment of Q fever, Antimicrobial Agents Chemother. 37 (1993) 1733–1736. [25] Marrie T.J., Stein A., Janigan D., Raoult D., Route of infection determines the clinical manifestations of acute Q fever, J. Infect. Dis. 173 (1996) 484–487. [26] LaScola B., Lepidi H., Raoult D., Pathologic changes during acute Q fever: influence of the route of infection and inoculum size in infected guinea pigs, Infect. Immun. 65 (1997) 2443–2447. [27] Hackstadt T., Williams J.C., Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii, Proc. Natl. Acad. Sci. USA 78 (1981) 3240–3244. [28] Amano K.I., Williams J.C., Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii, J. Bacteriol. 160 (1984) 994–1002. [29] Moos A., Hackstadt T., Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model, Infect. Immun. 55 (1987) 1144–1150. [30] Amano K., Williams J.C., Missler S.R., Reinhold V.N., Structure and biological relationships of Coxiella burnetii lipopolysaccharides. J. Biol. Chem. 262 (1987) 4740–4747. [31] McCaul T.F., Williams J.C., Developmental cycle of Coxiella burnetii: structure and morphogenesis of vegetative and sporogenic differentiation, J. Bacteriol. 147 (1981) 1065–1076. [32] Heinzen R.A., Hackstadt T., Samuel J.E., Developmental biology of Coxiella burnetii. Trends Microbiol. 7 (1999) 149–153. [33] McCaul T.F., Dare A.J., Gannon J.P., Galbraith A.J., In vivo endogenous spore formation by Coxiella burnetii in Q fever endocarditis, J. Clin. Path. 47 (1994) 978–981. [34] Oswald W., Thiele D., A sporulation gene in Coxiella burnetii?, J. Vet. Med. B 40 (1993) 366–370. [35] Moulder J.W., Interaction of Chlamydia and host cells in vitro, Microbiol. Rev. 55 (1991) 49–190. [36] Baca O.G., Klassen D.A., Aragon A.S., Entry of Coxiella burnetii into host cells, Acta Virol. 37 (1993) 143–155. [37] Meconi S., Jacomo V., Bouquet P., Raoult D., Mege J.L., Capo C., Coxiella burnetii induces reorganization of the actin cytoskeleton in human monocytes, Infect. Immun. 66 (1998) 5527–5533. [38] Tujulin E., Macellaro A., Lilliehöök B., Norlander L., Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells, Acta Virol. 42 (1998) 125–131. [39] Thompson H.A., Bolt C.R., Hoover T., Williams J.C., Induction of heat-shock proteins in Coxiella burnetii, New York Acad. Sci. 590 (1990) 127–135. 423
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[40] Vodkin M.H., Williams J.C., A heat shock operon in Coxiella burnetii produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli, J. Bacteriol. 170 (1988) 1227–1234. [41] Macellaro A., Tujulin E., Hjalmarsson K., Norlander L., Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii, Infect. Immun. 66 (1998) 5882–5888. [42] Zügel U., Kaufmann S.H.E., Role of heat shock proteins in protection from and pathogenesis of infectious diseases, Clin. Microbiol. Rev. 12 (1999) 19–39. [43] Zuber M., Hoover T.A., Court D.L., Cloning, sequencing and expression of the dnaJ gene of Coxiella burnetii, Gene 152 (1995) 99–102. [44] Zuber M., Hoover T.A., Court D.L., Analysis of a Coxiella burnetii gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC, J. Bacteriol. 177 (1995) 4238–4244. [45] Redd T., Thompson H.A., Secretion of proteins by Coxiella burnetii, Microbiology 141 (1995) 363–369. [46] Mo Y.Y., Cianiotto N.P., Mallavia L.P., Molecular cloning of a Coxiella burnetii gene encoding a macrophage infectivity potentiator (Mip) analogue, Microbiology 141 (1995) 2861–2871. [47] Mo Y., Seshu J., Wang D., Mallavia L.P., Synthesis in Escherichia coli of two smaller enzymatically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene, Biochem. J. 335 (1998) 67–77. [48] Mo Y., Mallavia L.P., A Coxiella burnetii gene encodes a sensor-like protein, Gene 151 (1994) 185–190. [49] Baca O.G., Roman M.J., Glew R.H., Christner R.F., Buhler J.E., Aragon A.S., Acid phosphatase activity in Coxiella burnetii: a possible virulence factor, Infect. Immun. 61 (1993) 4232–4239. [50] Akporiaye E.T., Baca O.G., Superoxide production and superoxide dismutase and catalase activities in Coxiella burnetii, J. Bacteriol. 154 (1983) 520–523. [51] McCaul T.F., The developmental cycle and Coxiella burnetii in: Williams J.C., Thompson H.A. (Eds.), Q Fever: The Biology of Coxiella burnetii, CRC Press, Boca Raton, Florida, USA, 1991, pp. 223–258. [52] Willems H., Jäger, C., Baljer, G., Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii, J. Bacteriol. 180 (1998) 3816–3822. [53] Afseth G., Mallavia L.P., Copy number of the 16S rRNA gene in Coxiella burnetii, Eur. J. Epidemiol. 13 (1997) 729–731. [54] Samuel J.E., Frazier M.E., Kahn M.L., Thomashow L.S., Mallavia L.P., Isolation and characterisation of a plasmid from phase I Coxiella burnetii, Infect. Immun. 41 (1983) 488–493. [55] Thiele D., Willems H., Haas M., Krauss H., Analysis of the entire nucleotide sequence of the cryptic plasmid QpH1 from Coxiella burnetii, Eur. J. Epidemiol. 10 (1994) 413–420.
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Norlander
[56] Valková D., Kazár J., A new plasmid (QpDV) common to Coxiella burnetii isolates associated with acute and chronic Q fever, FEMS Microbiol. Lett. 125 (1995) 275–280. [57] Willems H., Ritter, M., Jäger, C., Thiele, D., Plasmidhomologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217, J. Bacteriol. 179 (1997) 3293–3297. [58] Jäger C., Willems H., Thiele D., Baljer G., Molecular characterization of Coxiella burnetii isolates, Epidemiol. Infect. 120 (1998) 157–164. [59] Hall W.C., White J.D., Kishimoto R.A., Whitemire R.E., Aerosol Q fever infection of the nude mice, Vet. Pathol. 18 (1981) 672–683. [60] Scott G., Williams J.C., Stephenson E.H., Animal models in Q fever: Pathological responses of inbred mice to phase I Coxiella burnetii, J. Gen. Microbiol. 133 (1987) 691–700. [61] Khavkin T., Tabibzadeh S.S., A histologic, immunofluorescence and electron microscopic study of infectious process in mouse lung after intranasal challenge with Coxiella burnetii, Infect. Immun. 56 (1988) 1792–1799. [62] Paretsky D., The biology of Coxiella burnetii and the pathobiochemistry of Q fever and its endotoxicosis, NY Acad. Sci. 590 (1990) 416–421. [63] Kaufmann S.H.E., Immunity to intracellular microbial pathogens, Immunol. Today 16 (1995) 338–342. [64] Mege J.L., Maurin M., Capo C., Raoult D., Coxiella burnetii: the “query” fever bacterium. A model of immune subversion by a strictly intracellular microorganism, FEMS Microbiol. Rev. 19 (1997) 209–217. [65] Hackstadt T., Steric hindrance of antibody binding to surface proteins of Coxiella burnetii by phase I lipopolysaccharide, Infect. Immun. 56 (1988) 802–807. [66] Vishwanath S., Hackstadt T., Lipopolysaccharide phase variation determines the complement-mediated serumsusceptibility of Coxiella burnetii, Infect. Immun. 56 (1988) 40–44. [67] Turco J., Thompson H.A., Winkler H.H., Interferon-c inhibits growth of Coxiella burnetii in mouse fibroblasts, Infect. Immun. 45 (1984) 781–783. [68] Dellacasagrande J., Capo C., Raoult D., Mege J-L., IFN-γmediated control of Coxiella burnetii survival in monocytes: The role of cell apoptosis and TNF, J. Immunol. 162 (1999) 2259–2265. [69] Capo C., Amirayan N., Ghigo E., Raoult D., Mege J.L., Circulating cytokine balance and activation markers of leukocytes in Q fever, Clin. Exp. Immunol. 115 (1999) 120–123. [70] Tujulin E., Lilliehöök B., Macellaro A., Sjöstedt A., Norlander L., Early cytokine induction in mouse P388D1 macrophages infected by Coxiella burnetii, Vet. Immunol. Immunopathol. 68 (1999) 159–168. [71] Izzo A.A., Marmion B.P., Variation in interferon-gamma response to Coxiella burnetii antigens with lymphocytes from vaccinated or naturally infected subjects, Clin. Exp. Immunol. 94 (1993) 507–515.
Microbes and Infection 2000, 417-424
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Q fever epidemiology and pathogenesis Lena Norlander Defence Research Establishment, Division of NBC Defence, SE-901 82 Umeå, Sweden
ABSTRACT – The lungs are a port of entry and primary infectious focus of Coxiella burnetii, the obligate intracellular contagium of the worldwide zoonosis Q fever. The infectious process and immune response are characterised by studies in cell culture and animal systems. Following endocytosis, replication exclusively occurs in the phagolysosome. Several potential virulence factors are described. © 2000 Éditions scientifiques et médicales Elsevier SAS Q fever / Coxiella burnetii / epidemiology / pathogenesis / host response
1. Introduction
2. Epidemiology
Q fever is a worldwide zoonosis caused by Coxiella burnetii, an obligatory intracellular organism which is a member of the family Rickettsiaceae [1]. Recently, the use of molecular biology techniques has shown that C. burnetii is phylogenetically distant from the other members of this family [2]. C. burnetii belongs to the gamma subdivision of the Proteobacteria. C. burnetii is widely distributed in nature and responsible for infection in various mammals, including humans, and in birds, reptiles and fish [3, 4]. Humans beings are, however, the only host known to develop illness as a result of infection. Infection in animals is mainly subclinical but has been associated with late abortions, stillbirth, delivery of weak offspring and also infertility [5]. The microorganism is maintained in nature by two cycles that are essentially independent but occasionally overlapping. The basic cycle involves many species of wildlife and their ectoparasites, and the second cycle domestic animals [5]. Ticks are considered to be the natural primary reservoirs of C. burnetii responsible for the spread of the infection in wild animals and for transmission of C. burnetii from wild to domestic animals. Among domestic animals, cattle, sheep and goats are considered to be the main reservoirs of the agent responsible for infection of humans. Infected female animals shed large numbers of C. burnetii through their placenta and birth fluid, colostrum and milk into the environment. The excretions are potential sources of infection in animals and humans via inhalation of infectious aerosol of airborne dust. Estimates of infectivity for humans range from 1 to 10 bacteria. Since C. burnetii is very resistant and little affected by extreme environmental conditions, it can form a highly infectious dust and it remains viable over long period of time [5].
In 1955 Q fever was known to occur in 51 countries on five continents, and in the last decade the disease had been reported in yet another 8 countries [6]. Furthermore, seroepidemiological surveys have indicated the presence of Q fever in several other countries. New Zealand remains probably the only country in the world where Q fever is absent [7]. There is a remarkable diversity in the epidemiology of Q fever in different geographical regions. In most European countries Q fever attracts relatively low attention because of the low incidence of disease [5, 6]. The outbreaks during the last decade have usually been of limited size and in many cases the source of the Q fever infection is not identified. The largest outbreak of acute Q fever in the UK occurred in 1989. The most likely route of infection for the 147 diagnosed cases was assumed to be windborne spread from farmland to an urban area. Sheep and cattle in the UK are endemically infected with C. burnetii [8]. In northern Italy an increased number of Q fever cases was correlated to indirect exposure to sheep, i.e., by aerosol exposure to the infectious agent in the neighbourhood of farms [9]. A study of a German Q fever outbreak also suggested airborne transmission of C. burnetii from a large sheep farm after lambing season [10]. The results demonstrated that many residents were infected but had minimal or no symptoms. France has a high prevalence of acute Q fever, which is assumed to reflect a high national awareness of the disease. Chronic Q fever is also more frequently reported from France than other countries [11]. In contrast, in the former Czechoslovakia and the present Slovakia, several hundred cases of acute Q fever have been reported during the last two decades, but no case of chronic Q fever has been confirmed. The correlation between infected domes-
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tic animals and Q fever has been established, i.e., the source of a Q fever outbreak in Slovakia affecting more than a hundred persons was shown to be imported goats [12]. In Bulgaria, goats have been concluded to be the main reservoir for C. burnetii in recent years [13]. Hundreds of serologically confirmed cases of acute Q fever have occurred and chronic forms of the disease have also been observed. Studies of the epizootiological and epidemiological situation of Q fever in Poland indicated C. burnetii to be common among sheep and cattle, but few human cases have been reported [14]. Extremely few cases of domestic Q fever have been diagnosed in Sweden, but the contagium has been isolated from sheep placentas and from mouldy hay ( [15], Macellaro, unpublished data). A recent survey of Q fever showed that sheep farmers and veterinarians were risk groups for exposure to C. burnetii [16]. Similar observations, i.e., a strong correlation between domestic animals and limited epidemics/sporadic cases of Q fever, are reported also from other continents than Europe. Serosurveys on the occurrence of C. burnetii in central and eastern Canada, showed a high prevalence of antibodies to the contagious agent in blood donors [17]. In one of these regions no case of Q fever had never been reported, while sporadic cases appeared in the other region. Russia has Q fever with foci of different intensity in large cattle and sheep breeding complexes and farms and also in poultry factories [18]. But there is also a high prevalence of antibodies to C. burnetii among individuals that have no professional contact with animal or animal products, which indicates a change in the epidemiological patterns of Q fever [19]. The infection is widespread in Japan, where domestic animals have been suggested to be important reservoirs of C. burnetii [20].
3. C. burnetii infection in humans Human infection with C. burnetii can be either subclinical, acute or chronic [21]. Asymptomatic Q fever infection is common, since the number of serologically positive persons is much higher than the incidence rate of the disease [5]. The incubation time for acute Q fever ranges from two to four weeks. In acute Q fever the symptoms resemble those of influenza (fever, headache and myalgia), and the disease may become complicated with pneumonia and/or granulomatous hepatitis. Cases of febrile reruptions, myocarditis, pericarditis and meningoencephalitis have been reported [21]. C. burnetii has been isolated from placenta and breast milk from mothers who have had an acute Q fever infection a few years before delivery. This indicates that resting C. burnetii undergoes reactivation during pregnancy. Some of the mothers gave birth to healthy children, while others suffered miscarriages and delivery of abnormal children [21]. Chronic Q fever, which is most often characterized by endocarditis, may develop years after an acute infection. In contrast to acute Q fever, the mortality rate for chronic Q fever has been reported to be high [21]. The development of acute or chronic Q fever infection is assumed to depend on the patient’s condition and 418
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immune status rather than on specific ’acute’ or ’chronic’ C. burnetii strains [22, 23]. If acute Q fever is diagnosed, antibiotic treatment is recommended, but the disease usually also resolves without treatment. Tetracycline compounds, especially doxycycline and also quinolone compounds are currently used to treat Q fever. In chronic Q fever, treatment with a combination of several antibiotics lasting from one to several years is necessary [21, 24]. Experimental infection of animals indicates that the route of infection determines the predominant manifestations of the acute form of the disease [25, 26]. Intraperitoneal inoculation of C. burnetii in guinea pigs led to pathologic changes mainly in the liver, whereas intranasal infection caused pathologic changes mainly in the lungs. The size of the inoculum influenced clinical expression in acute Q fever [26]. A correlation between route of infection and the predominant symptoms could explain the variation in manifestations of acute Q fever in different geographical areas. In southeast Canada, Switzerland and northern Spain the main manifestation of acute Q fever is pneumonia, assumed to be caused by inhalation of contaminated aerosols. In contrast, in France or southern Spain it is granulomatous hepatitis which has been speculated is due to caused by ingestion of contaminated raw milk [26].
4. The contagious agent of Q fever The aetiological agent of Q fever, C. burnetii, is an obligate intracellular bacterium and during the infectious process, it is taken up by host cells into phagosomes, which then fuse with primary lysosomes to form phagolysosomes. C. burnetii is unique among the pathogenic bacteria because it has evolved biochemical strategies of growth in the hostile acidic phagolysomes of host cells. Actually, the acidic content of the phagolysosome is a requisite for its growth and multiplication [27]. C. burnetii has a cell wall similar to that of Gramnegative bacteria and it is characterised by a phase transition [1, 28]. C. burnetii phase I, which is isolated from infected humans or animals, has a smooth hydrophilic lipopolysaccharide (LPS) surface structure. Upon cultivation in tissue cell cultures or in embryonated hen’s eggs, this smooth type of LPS is lost and the surface of the phase II variant contains truncated rough-type LPS molecules with a hydrophobic surface. The phase transition is accompanied by a loss of virulence [29]. A mutant with an LPS of intermediate complexity has been described. The LPS of C. burnetii has an endotoxic activity which is 100 to 1 000 times less than the LPS of Enterobacteria [30]. The extreme stability of C. burnetii has been associated with the small dense cell forms and spore-like forms observed in electron microscopy studies [31]. C. burnetii has a developmental cycle which consists of both vegetative growth by binary fission and unequal cell division leading to spore-like formation. This cycle results in the formation of a heterogeneous mixture of pleomorphic cell types with morphologically distinguishable cell variants. The compact small cell variants (SCV) (0.2–0.5 µm) are Microbes and Infection 2000, 417-424
Q fever epidemiology and pathogenesis
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Figure 1. Electron micrograph of a Buffalo green monkey cell heavily infected by C. burnetii.
metabolically dormant and they are assumed to survive extracellularly for long periods. In contrast, the large cell variants (LCV) (≥1.0 µm) have a high metabolic activity and have lost the resistant properties. Both small and large cell variants are infectious in in vitro and in vivo models. Several proteins that are differentially synthesized by the two variants have been characterized and these are described in a recent review [32]. The spore-like forms have been observed in the large cell variants and the formation is thought to occur by a process that resembles asymmetric cell division [31]. Spore-like formation has also been detected in C. burnetii-infected cardiac valves [33]. Moreover, a partial sequence of a sporulation gene in C. burnetii has recently been published [34]. Spore particles have, however, not yet been prepared in pure form.
5. Entry into and proliferation in host cells C. burnetii infects a great number of cell types including monocytes, macrophages and a variety of transformed cells [1]. The ability to efficiently invade and subsequently grow within eukaryotic cells is a key virulence factor for organisms, which require an intracellular environment for their multiplication. C. burnetii could be expected to utilize several cell processes for uptake – endocytosis – in a similar way to that of the obligate intracellular species of Chlamydiaecae [35]. Several publications have reported that C. burnetii reaches its internal niche by phagocytosis and it enters passively [1, 36]. The phase II bacteria are more readily internalised by the host cells than are the phase I variants. Opsonisation by specific Ig enhances phagocytosis of both phase I and II C. burnetii. Moreover, based on recent observations, additional variants of endocytosis have been suggested to be used in C. burnetii internalisation. Meconi et al. [37] showed that virulent, but not avirulent, C. burnetii stimulated morphological Microbes and Infection 2000, 417-424
changes in monocytes similar to those observed in macropinocytosis of Salmonella typhimurium. Upon contact between C. burnetii and the host cell membrane, protrusions and polarized projections are induced over an extensive region of the cell surface. In addition, studies on the efficient internalisation of the phase II variants into macrophages after treatment by inhibitors of endocytosis indicate that pinocytic pathways are involved [38]. The simulataneously use of various endocytic variants for its internalisation in a wide spectrum of host cells offers an advantage for C. burnetii in establishing infection. C. burnetii is a slowly growing bacterium with a generation time of several hours. Studies of C. burnetii growth in tissue cell cultures show proliferation of bacteria in vacuoles which may fuse and form a huge single vacuole. This vacuole can occupy most of the enlarged cell’s volume and the nucleus and cytoplasm are displaced to the outermost part of the infected cell. In spite of the bacterial burden, such a cell seems to grow almost normally [1] (figure 1).
6. Virulence factors and their genes Though C. burnetii has been recognised worldwide as an important pathogen, there is limited knowledge of its virulence genes. The functions and activities of proteins on the C. burnetii cell surface and proteins exported to the exterior growth environment are likely to be important for understanding aspects of its intracellular survival and growth. C. burnetii protein synthesis can be studied in a cell-free acidic system, where nucleic acid and protein synthesis as well as a minor increase in cell size are demonstrated [39]. Growth, however, by the definition doubling of both mass and number of cells, does not occur in this in vitro system. Incubation of C. burnetii in the acidic in vitro system rapidly triggers the synthesis of a number of proteins [39]. This in vitro activation of protein synthesis is assumed to 419
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reflect the initial production of proteins in vivo and thus, is a tool to study proteins of significance for the early phase of infection. Among these proteins are heat shock protein 60 and 70 homologues, Hsp62 and Hsp71 [40, 41]. The Hsp62 gene htpB is colocalized with htpA. The genes htpA and htpB correspond to the groES and groEL of Escherichia coli [40]. The Hsp70 homologue in C. burnetii is the hsp71 gene product, a protein with high homology to the E. coli DnaK protein [41]. Both htpB and hsp71 are under the regulation of a promoter with a strong sequence homology to the E. coli consensus heat shock promoter. Hsp71 has been shown to be cell wallassociated in addition to the cytosolic localization expected for chaperones [41]. It is not known if the dual localization in C. burnetii reflects dual roles of Hsp71. Additional functions of chaperons of the Hsp60 and Hsp70 families of other pathogens have been suggested, i.e., invasion in cell culture models. Several prokaryotic heat shock proteins have also been shown to be important immunogens and to stimulate both T cells and B cells [42]. Both the 62- and 71-kDa Hsp of C. burnetii are reported to be immunodominant [40, 41]. A third heat shock protein gene, dnaJ, has also been described in C. burnetii [43]. Another C. burnetii protein, the mucZ gene product, shares C-terminal sequence homology with the J domain of the DnaJ protein, and it was demonstrated to induce capsule synthesis in E. coli [44]. Potential interactions are suggested between MucZ and the DnaK-chaperon machinery and a requirement for DnaK is indicated. The observed surface localization of Hsp71 may indicate a role of the protein delivery of the mucZ gene product to the surface. A possible virulence property of C. burnetii is the demonstrated active secretion of proteins. In vitro studies suggest that specific secretion of selected proteins from the bacteria occurs a few hours after exposure to an acidic environment [45]. This might reflect a mechanism for efficient delivery of selected bacterial proteins to the interior of the host cell, thus opening a possibility for the bacterium to ’communicate’ with its host. In Q fever, macrophages play a central role, since they are host cells and involved in the dissemination of C. burnetii. By looking for sequences of potential virulence compounds of other pathogens with similar infection niches, a number of C. burnetii genes have been isolated. One example is the macrophage infectivity potentiator gene, cbmip [46]. Mip has been demonstrated in several intracellular pathogens such as Legionella pneumophila and Chlamydia trachomatis, where it has been shown to have a role as a virulence factor. The enzymatic activity of Mip proteins (PPIase) appears to be related to the ability of intracellular organisms to initiate infection. Further characterization of cbmip revealed a mechanism to optimise the utilization of the limited genome capacity of C. burnetii (about one third of the E. coli genome). Mo et al. identified two additional proteins, which were synthesized from the same open reading frame, but starting at different internal translation start codons [47]. All three proteins exhibit similar PPIase activities. The product of C. burnetii qrsA (Q fever agent regulatory sensor-like gene) has also been identified as a homo420
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logue to a virulence gene of pathogens [48]. QrsA is a PhoQphoR homologue, which is involved in regulation of acid phosphatase gene expression in Salmonella. C. burnetii has been shown to produce acid phosphatase activity which could account for its observed capacity to inhibit the oxidative burst in neutrophils [49]. Moreover, C. burnetii possesses superoxide dismutase and catalase, which confer protection from the microbicidal effects of reactive oxygen intermediates of the host cells [50]. The factors involved in the bacterial differentiation confer a flexibility of great value for C. burnetii. A number of proteins, which are differentially expressed in SCV and LCV, have been described [32, 51]. ScvA and Hq1 are SCV-specific proteins that are localized to the cytoplasm. These proteins are assumed to have structural roles in the formation of the condensed SCV nucleoid. They are degraded during development of the SCV to the LCV. The outer membrane proteins OMP34 and P1 are differentially synthesized in the two cell variants. At least two other differentially expressed proteins have been identified, i.e., homologues to the elongation factors (EF) EF-Tu and EF-Ts.
7. Genome and plasmids of C. burnetii Recently, a physical and genetic map was constructed of the 2 103-kb C. burnetii linear chromosome [52]. Open reading frames with homology to database entries and genes were located on the physical map by southern hybridization. The slow growth of C. burnetii is assumed to be a reflection of the single rRNA operon copy, since the presence of a low copy number of the rRNA operon is a trend among slow-growing bacteria, including other obligate intracellular bacteria [53]. Several authors have reported the existence of cryptic plasmids in C. burnetii. Initial studies identified the first C. burnetii plasmid, the 36-kb QpH1, from the American isolate Nine Mile [54]. The entire nucleotide sequence of this plasmid has been deduced [55]. Screening of a large number of isolates from different geographic sources has also shown the existence of other plasmids in C. burnetii. The larger QpRS (38–39 kb) and QpDG (51 kb) as well as the slightly smaller QpDV plasmids and QpH1 have 25 to 39-kb regions, which share a large amount of homology [55, 56]. These sequences are also present in the chromosome of a plasmidless C. burnetii isolate [57]. Comparison of the entire QpH1 plasmid sequence of phase I and II, and plasmid sequences integrated into the chromosome of a plasmidless strain phase I and II, revealed no evidence for specific genes or sequences involved in phase variation [55, 57]. Furthermore, the general view today is that there is no correlation between plasmid type of the infectious agent and acute or chronic form of the disease [22, 23]. Moreover, genetic characterisation of isolates from patients with acute and chronic Q fever demonstrated a considerable heterogeneity between the isolates, and they could not be grouped into acute and chronic strains. Restriction fragment length polymorphism was used to differentiate 80 C. burnetii isolates from various geographical origins. This revealed 20 different restriction Microbes and Infection 2000, 417-424
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groups and evolutionary relationships among groups, which corresponded to the geographical origin of the isolates [58].
8. Infection models of Q fever The predominant form of the disease is the acute form of Q fever. During an acute Q fever infection any organ may be involved, but the lungs and liver are most often affected. The lungs not only serve as a portal of entry of the pathogen into the human body, but also an area of primary infectious focus. In the lung, the principal defensive mechanism is the clearance of the particulate microorganisms by either the mucociliary process in conducting airways or the transport of the microorganisms from the lung to the regional tracheobronchial lymph nodes. The mobile lung macrophages may participate in the systemic spread of C. burnetii by retaining them in their intracellular acidic vesicles. The most common animal model systems for studies of Q fever are guinea pigs and mice. The mouse model of Q fever is attractive to use for studies of airborne infection of C. burnetii since there is a considerable similarity among mammalian species in overall lung composition and in the distribution and features of cells of the respiratory system. There are several publications concerning experimental intranasal or aerosol infection of mice with C. burnetii [59-61]. Symptoms of disease are described as lethargy, ruffled fur and, in some sensitive mice strains, even death. Pneumonia and hepatitis may also be observed. The infectious process in lungs of sensitive mice has been described as either interstitial or intra-alveolar inflammation with the macrophages prevailing in the excudate. There is proliferation of bronchiolar epithelium and desquamation of cells. The infection is self-limiting with a complete resolution and also associated with the influx of T cells in the pneumonic foci. In mouse liver focal mononuclear cell aggregates are observed. Gross lesions consisting of enlargement of spleen have been reported one to two weeks postinfection. In guinea pigs infected by C. burnetii the gross pathology of liver has a characteristic pattern such as pyrexia, development of hepatomegaly and fatty infiltration [62]. Studies on the pathobiochemical events and their regulation during Q fever in guinea pigs showed specific effects on hepatic metabolic processes. Stimulated hepatic transcription and translation of certain RNA and protein species attend the development of the disease, as do phosphorylation and dephosphorylation of central RNA and protein species.
9. Innate and acquired immunity Immunity to intracellular microbial pathogens is assumed to be essentially due to cell-mediated defence development [63]. The successful elimination of such pathogens depends on the activation of the appropriate blend of T cells and resistance correlates with a Th1dominated immune response. There is a close interaction Microbes and Infection 2000, 417-424
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between the innate and acquired immune response, i.e., the divergence into Th1 and Th2 cells is regulated by the innate immune system. Macrophages have important immunoregulatory functions in development of protective immunity. Proinflammatory cytokines such as interleukin 1 (IL-1), tumour necrosis factor (TNF) and interleukin 12 (IL-12), which are secreted by infected macrophages, are capable of modulating the cellular immune response. Gene-knockout studies of cytokines and their receptors have verified the essential role of interferon γ (IFN-γ) and TNF in defence against intracellular pathogens [63]. A number of observations verify the requirement of cell-mediated response for clearance of C. burnetii infection also. It has been shown that infection of athymic mice (lacking T cells) by C. burnetii produces a progressive nonlethal disease, while normal mice cleared the infection in a few weeks [59]. Moreover, chronic Q fever is developed by humans with defective immunological status and T-cell-mediated immunity is depressed in Q fever endocarditis [64]. The role of antibodies in Q fever is controversial, but most investigators support the view that antibodies are not required for control of C. burnetii infection. Antibodies, however, may contribute to the elimination of C. burnetii, i.e., in the immune host, professional phagocytes are influenced by antibodies. Moreover, C. burnetii phase II organisms are more exposed for binding of opsonising antibodies than are phase I bacteria, whose LPS masks surface antigens from the immune system [65]. The preferential killing of avirulent phase II bacteria is also suggested to be due to binding of complement C3b [66]. Phase I organisms resist complement-mediated serum killing and this appears to be correlated with the LPS. In vitro and in vivo studies demonstrate that IFN-γ and TNF play important roles in the elimination of C. burnetii [67-69]. Growth of C. burnetii in a monocytic cell line was inhibited by IFN-γ and this cytokine also induced apoptosis of infected monocytes [67]. IFN-γ-mediated killing of C. burnetii and death of infected monocytes were both dependent on TNF. Macrophages infected in vitro by virulent C. burnetii had an early and strong induction of TNF-α [70]. The triggering of TNF-α was equivalent in cells infected by C. burnetii phase I and phase II. C. burnetii in macrophages would have a survival advantage by downregulating the IFN-γ production, which otherwise would activate the host cell. In vitro studies have shown that the IFN-γ production is inhibited when C. burnetii phase I LPS interacts with monocytes and macrophages [71]. Modification of phase I LPS sugar chains to form an artificial phase II LPS sharply increases IFN-γ when exposed to the test system, even though the protein composition remains the same [71]. These findings are in agreement with the observation of experimental infection caused by C. burnetii phase I and phase II, respectively. The phase II bacteria are more readily eliminated and this might in part be due to a less efficient restriction of the IFN response. The phase I-infected macrophages also produced IL-1α. In contrast, no similar IL-1α induction was found in macrophages infected by avirulent phase II bacteria. The 421
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differential induction of IL-1α by the two variants could not be correlated to LPS purified from phase I or phase II C. burnetii, respectively [70]. The early IL-1α induction in macrophages infected by C. burnetii phase I, but not phase II, indicates a specific role of the cytokine in the infectious process. When the levels of circulating inflammatory cytokines and their antagonists were determined in leucocytes of Q fever patients, the level of TNF was found to be markedly increased [69]. The high TNF level was most pronounced in blood from patients with chronic Q fever (endocarditis), while the levels in acute Q fever cases were lower. Moreover, the increased TNF levels are assumed to result in the observed upregulation of TNF receptor type II. There was also increased levels of IL-1 receptor antagonist, which is known to block the activity of IL-1 [69]. This may contribute to a decreased resistance to C. burnetii infection.
10. Conclusion Q fever attracts relatively low attention in most countries because of the moderate disease incidence rate and the limited size of the outbreaks. It is often impossible to identify the source of infection, but domestic animals are considered to be the main reservoirs of C. burnetii responsible for infection of humans. Characterisation of isolates from various geographical sources revealed evolutionary relationships among groups that correspond to the geographical origin of the isolate. The development of human infection, which is either acute or chronic, is correlated to the patient’s condition and immune status rather than to specific ’acute’ or ’chronic’ C. burnetii strains. In acute Q fever, which is the most common manifestation of the disease, the symptoms resemble those of influenza. The route of infection, i.e., by aerosol or oral, determine the predominant symptoms. Chronic Q fever is most often characterized by endocarditis and it may develop years after an acute infection. Acute Q fever usually resolves without treatment, while chronic infection requires prolonged treatment with a combination of several antibiotics. In vitro and in vivo studies demonstrate that IFN-γ and TNF play important roles in the host defence processes leading to the elimination of C. burnetii. Recent observations also indicate the involvement of IL-1. Cell-mediated immune response is required for clearance of a C. burnetii infection and T cell-mediated immunity is shown to be depressed in Q fever endocarditis. Moreover, data on circulating inflammatory cytokines and receptor antagonists suggest a shift of cytokine balance towards cytokine antagonists in acute Q fever. This may contribute to a decreased resistance to C. burnetii infection. C. burnetii is characterised by a phase transition phenomenon involving LPS. The phase transition is accompanied by a loss of virulence. So far, the LPS is the only component verified to differ between the virulent phase I and avirulent phase II of C. burnetii. The bacterium has an extreme stability, which has been explained by the different morphological forms of its developmental cycle, i.e., the non-metabolic SCV and the endospore-like forms. The 422
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metabolically active LCV has lost the resistant properties. Both cell variants are infectious in vitro and in vivo. Several proteins that are differentially synthesized by the two cell variants have been characterized. It is to be assumed that these proteins are important contributions to the ability of C. burnetii to act as an efficient contagious agent. C. burnetii has an unique adaptation to the harsh acidic milieu of the phagolysosomes, to which it is delivered after endocytosis. Recent reports indicate the use of various endocytic pathways for efficient internalisation, which should be a prerequisite for a successful obligate intracellular bacterium. Physical and genetic maps have been constructed for the 2 103-kb C. burnetii linear chromosome as well as for the crypic plasmid variants, which share large regions of homology. There is no evidence for specific genes or sequences involved in the phase transition or in disease manifestation. The absolute requirement for low pH has been utilised for in vitro studies of protein induction and these studies have demonstrated the production of several Hsp being essential proteins and potential virulence factors, i.e., homologues to E. coli GroEL, GroES, DnaK and DnaJ. Screening of gene libraries for other potential virulence factors, such as factors required for intracellular survival, has successfully demonstrated a number of such proteins. Examples are three slightly different macrophage potentiator factors and a regulatory sensor-like protein which is suggested to have a role in regulation of acid phosphatase. The acid phosphatase may account for inhibition of the intracellular bactericidal oxidative burst. Moreover, C. burnetii possesses superoxide dismutase and catalase, which confer protection from the microbiocidal effects of reactive oxygen intermediates of the host cells. Taken together, however, there are so far few ’virulence factors’ described for C. burnetii. This is assumed to be due to the problems concerning studies on intracellular organisms. For the future, genome sequencing and the recently described genetic system for C. burnetii are promising tools for studies to increase the knowledge of the pathogenic nature of this organism.
Acknowledgments I am grateful to Karin Hjalmarsson and Anna Macellaro for helpful discussions and critical reading of the manuscript and to Lenore Johansson for the electron microscopic photo.
References [1] Baca O.G., Paretsky D., Q fever and Coxiella burnetii: a model for host-parasite interactions, Microbiol. Rev. 46 (1983) 127–149. [2] Stein A., Saunders N.A., Taylor A.G., Raoult D., Phylogenic homogenicity of Coxiella strains as determined by 16S ribosomal RNA sequencing. FEMS Microbiol. Lett. 113 (1993) 339–244. Microbes and Infection 2000, 417-424
Q fever epidemiology and pathogenesis
[3] Sawyer L.A., Fishbein D.B., McDade J.E., Q fever – current concepts, Rev. Infect. Dis. 9 (1987) 935–946. [4] Rehácek J., Tarasevich I.V., IV Q fever, in: Gresiková M. (Ed.), Acari-borne rickettsiae and rickettsioses in Eurasia, VEDA, Bratislava, 1988, pp. 203–243. [5] Aitken I.D., Bögel K., Cracea E., Edlinger E., Houwers D., Krauss H., Rády M., Rehácek J., Schiefer H.G., Schmeer N., Tarasevich I.V., Tringali G., Q fever in Europe: Current aspects of aetiology, epidemiology, human infection, diagnosis and therapy, Infection 15 (1987) 323–327. [6] Kazár J., Q fever – current concept, in: Raoult D., Brouqui P. (Eds.), Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium, Elsevier, Amsterdam, 1999, pp. 304–319. [7] Hilbink F., Penrose M., Kovácová E., Kazár J., Q fever is absent from New Zealand, Int. J. Epidemiol. 22 (1993) 945–949. [8] Smith D.L., Ayres J.G., Blair I., Burge P.S., Carpenter M.J., Caul E.O., Couplands B., Desselberger U., Evans M., Farrell I.D., Hawker J.I., Smith E.G., Wood M.J., A large Q fever outbreak in the West Midlands: clinical aspects, Resp. Med. 87 (1993) 509–516. [9] Selvaggi T.M., Rezza G., Scagnelli M., Rigoli R. et al., Investigation of a Q fever outbreak in Northern Italy, Eur. J. Epidemiol. 12 (1996) 403–408. [10] Lyytikäinen O., Ziese T., Schwartländer B., Matzdorff P., Kuhnhen C., Jäger C., Petersen L., An outbreak of sheepassociated Q fever in a rural community in Germany, Eur. J. Epidemiol. 14 (1998) 193–199. [11] Raoult D., Q fever: still a query after all these years, J. Med. Microbiol. 44 (1996) 77–78. [12] Kovácová, E., Kazár J., Simková A., Clinical and serological analysis of a Q fever outbreak in western Slovakia with four-year follow-up, Eur. J. Clin. Microbiol. Dis. 17 (1998) 867–869. [13] Serbezov V., Kazár J., Novkirishki V., Gatcheva N., Kovácová E., Voynova V., Q fever in Bulgaria and Slovakia, Emerging Infect. Dis. 5 (1999) 388–394. [14] Anusz Z., Ciecierski H., Knap J., Piesiak Z., Platt-Samoraj A., Siemionek J., Szweda W., Szymamski M., Lecki J., Kowalska E., Q fever in Poland in the year 1952–1995, in: Kazár J., Toman R. (Eds.), Rickettsia and Rickettsial Diseases, Slovak Acad. Sci., Veda Bratislava, Slovakia, 1996, pp. 528–534. [15] Åkesson Å., Krauss H., Thiele H.D., Macellaro A., Schwan, O., Norlander, L., Isolation of Coxiella burnetii in Sweden, Scand. J. Infect. Dis. 23 (1991) 273–274. [16] Macellaro A., Åkesson Å., Norlander L., A survey of Q fever in Sweden, Eur. J. Epidemiol. 9 (1993) 213–216. [17] Marrie T.J., Seroepidemiology of Q fever in New Brunswick and Manitoba, Can. J. Microbiol. 34 (1988) 1043–1045. [18] Rudakov N.V., Ecologico-epidemiological observations of Q fever in Russia, in: Kazár J., Toman R. (Eds.), Rickettsia and Rickettsial Diseases, Slovak Acad. Sci., Veda Bratislava, Slovakia, 1996, pp. 524–527. [19] Rybakova N., Tokarevich N., Chaika N., Q fever monitoring in Vologda province in northern Russia, in: Raoult D., Brouqui P. (Eds.), Rickettsiae and Rickettsial Diseases at the Turn of the Third Millenium, Elsevier, Amsterdam, 1999, pp. 351–355. Microbes and Infection 2000, 417-424
Review
[20] Hirai K., To H., Advances in the understanding of Coxiella burnetii infection in Japan, J. Vet. Med. Sci. 60 (1998) 781–790. [21] Sawyer L.A., Fishbein D.B., McDade J.E., Q fever: current concepts, Rev. Infect. Dis. 9 (1987) 935–946. [22] Yu X., Raoult D., Serotyping Coxiella burnetii isolates from acute and chronic patients by using monoclonal antibodies, FEMS Microbiol. Lett. 117 (1994) 15–19. [23] Thiele D., Willems H., Is plasmid based differentiation of Coxiella burnetii in acute and chronic isolates still valid?, Eur. J. Epidemiol. 10 (1994) 427–434. [24] Raoult D., Treatment of Q fever, Antimicrobial Agents Chemother. 37 (1993) 1733–1736. [25] Marrie T.J., Stein A., Janigan D., Raoult D., Route of infection determines the clinical manifestations of acute Q fever, J. Infect. Dis. 173 (1996) 484–487. [26] LaScola B., Lepidi H., Raoult D., Pathologic changes during acute Q fever: influence of the route of infection and inoculum size in infected guinea pigs, Infect. Immun. 65 (1997) 2443–2447. [27] Hackstadt T., Williams J.C., Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii, Proc. Natl. Acad. Sci. USA 78 (1981) 3240–3244. [28] Amano K.I., Williams J.C., Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii, J. Bacteriol. 160 (1984) 994–1002. [29] Moos A., Hackstadt T., Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model, Infect. Immun. 55 (1987) 1144–1150. [30] Amano K., Williams J.C., Missler S.R., Reinhold V.N., Structure and biological relationships of Coxiella burnetii lipopolysaccharides. J. Biol. Chem. 262 (1987) 4740–4747. [31] McCaul T.F., Williams J.C., Developmental cycle of Coxiella burnetii: structure and morphogenesis of vegetative and sporogenic differentiation, J. Bacteriol. 147 (1981) 1065–1076. [32] Heinzen R.A., Hackstadt T., Samuel J.E., Developmental biology of Coxiella burnetii. Trends Microbiol. 7 (1999) 149–153. [33] McCaul T.F., Dare A.J., Gannon J.P., Galbraith A.J., In vivo endogenous spore formation by Coxiella burnetii in Q fever endocarditis, J. Clin. Path. 47 (1994) 978–981. [34] Oswald W., Thiele D., A sporulation gene in Coxiella burnetii?, J. Vet. Med. B 40 (1993) 366–370. [35] Moulder J.W., Interaction of Chlamydia and host cells in vitro, Microbiol. Rev. 55 (1991) 49–190. [36] Baca O.G., Klassen D.A., Aragon A.S., Entry of Coxiella burnetii into host cells, Acta Virol. 37 (1993) 143–155. [37] Meconi S., Jacomo V., Bouquet P., Raoult D., Mege J.L., Capo C., Coxiella burnetii induces reorganization of the actin cytoskeleton in human monocytes, Infect. Immun. 66 (1998) 5527–5533. [38] Tujulin E., Macellaro A., Lilliehöök B., Norlander L., Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells, Acta Virol. 42 (1998) 125–131. [39] Thompson H.A., Bolt C.R., Hoover T., Williams J.C., Induction of heat-shock proteins in Coxiella burnetii, New York Acad. Sci. 590 (1990) 127–135. 423
Review
[40] Vodkin M.H., Williams J.C., A heat shock operon in Coxiella burnetii produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli, J. Bacteriol. 170 (1988) 1227–1234. [41] Macellaro A., Tujulin E., Hjalmarsson K., Norlander L., Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii, Infect. Immun. 66 (1998) 5882–5888. [42] Zügel U., Kaufmann S.H.E., Role of heat shock proteins in protection from and pathogenesis of infectious diseases, Clin. Microbiol. Rev. 12 (1999) 19–39. [43] Zuber M., Hoover T.A., Court D.L., Cloning, sequencing and expression of the dnaJ gene of Coxiella burnetii, Gene 152 (1995) 99–102. [44] Zuber M., Hoover T.A., Court D.L., Analysis of a Coxiella burnetii gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC, J. Bacteriol. 177 (1995) 4238–4244. [45] Redd T., Thompson H.A., Secretion of proteins by Coxiella burnetii, Microbiology 141 (1995) 363–369. [46] Mo Y.Y., Cianiotto N.P., Mallavia L.P., Molecular cloning of a Coxiella burnetii gene encoding a macrophage infectivity potentiator (Mip) analogue, Microbiology 141 (1995) 2861–2871. [47] Mo Y., Seshu J., Wang D., Mallavia L.P., Synthesis in Escherichia coli of two smaller enzymatically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene, Biochem. J. 335 (1998) 67–77. [48] Mo Y., Mallavia L.P., A Coxiella burnetii gene encodes a sensor-like protein, Gene 151 (1994) 185–190. [49] Baca O.G., Roman M.J., Glew R.H., Christner R.F., Buhler J.E., Aragon A.S., Acid phosphatase activity in Coxiella burnetii: a possible virulence factor, Infect. Immun. 61 (1993) 4232–4239. [50] Akporiaye E.T., Baca O.G., Superoxide production and superoxide dismutase and catalase activities in Coxiella burnetii, J. Bacteriol. 154 (1983) 520–523. [51] McCaul T.F., The developmental cycle and Coxiella burnetii in: Williams J.C., Thompson H.A. (Eds.), Q Fever: The Biology of Coxiella burnetii, CRC Press, Boca Raton, Florida, USA, 1991, pp. 223–258. [52] Willems H., Jäger, C., Baljer, G., Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii, J. Bacteriol. 180 (1998) 3816–3822. [53] Afseth G., Mallavia L.P., Copy number of the 16S rRNA gene in Coxiella burnetii, Eur. J. Epidemiol. 13 (1997) 729–731. [54] Samuel J.E., Frazier M.E., Kahn M.L., Thomashow L.S., Mallavia L.P., Isolation and characterisation of a plasmid from phase I Coxiella burnetii, Infect. Immun. 41 (1983) 488–493. [55] Thiele D., Willems H., Haas M., Krauss H., Analysis of the entire nucleotide sequence of the cryptic plasmid QpH1 from Coxiella burnetii, Eur. J. Epidemiol. 10 (1994) 413–420.
424
Norlander
[56] Valková D., Kazár J., A new plasmid (QpDV) common to Coxiella burnetii isolates associated with acute and chronic Q fever, FEMS Microbiol. Lett. 125 (1995) 275–280. [57] Willems H., Ritter, M., Jäger, C., Thiele, D., Plasmidhomologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217, J. Bacteriol. 179 (1997) 3293–3297. [58] Jäger C., Willems H., Thiele D., Baljer G., Molecular characterization of Coxiella burnetii isolates, Epidemiol. Infect. 120 (1998) 157–164. [59] Hall W.C., White J.D., Kishimoto R.A., Whitemire R.E., Aerosol Q fever infection of the nude mice, Vet. Pathol. 18 (1981) 672–683. [60] Scott G., Williams J.C., Stephenson E.H., Animal models in Q fever: Pathological responses of inbred mice to phase I Coxiella burnetii, J. Gen. Microbiol. 133 (1987) 691–700. [61] Khavkin T., Tabibzadeh S.S., A histologic, immunofluorescence and electron microscopic study of infectious process in mouse lung after intranasal challenge with Coxiella burnetii, Infect. Immun. 56 (1988) 1792–1799. [62] Paretsky D., The biology of Coxiella burnetii and the pathobiochemistry of Q fever and its endotoxicosis, NY Acad. Sci. 590 (1990) 416–421. [63] Kaufmann S.H.E., Immunity to intracellular microbial pathogens, Immunol. Today 16 (1995) 338–342. [64] Mege J.L., Maurin M., Capo C., Raoult D., Coxiella burnetii: the “query” fever bacterium. A model of immune subversion by a strictly intracellular microorganism, FEMS Microbiol. Rev. 19 (1997) 209–217. [65] Hackstadt T., Steric hindrance of antibody binding to surface proteins of Coxiella burnetii by phase I lipopolysaccharide, Infect. Immun. 56 (1988) 802–807. [66] Vishwanath S., Hackstadt T., Lipopolysaccharide phase variation determines the complement-mediated serumsusceptibility of Coxiella burnetii, Infect. Immun. 56 (1988) 40–44. [67] Turco J., Thompson H.A., Winkler H.H., Interferon-c inhibits growth of Coxiella burnetii in mouse fibroblasts, Infect. Immun. 45 (1984) 781–783. [68] Dellacasagrande J., Capo C., Raoult D., Mege J-L., IFN-γmediated control of Coxiella burnetii survival in monocytes: The role of cell apoptosis and TNF, J. Immunol. 162 (1999) 2259–2265. [69] Capo C., Amirayan N., Ghigo E., Raoult D., Mege J.L., Circulating cytokine balance and activation markers of leukocytes in Q fever, Clin. Exp. Immunol. 115 (1999) 120–123. [70] Tujulin E., Lilliehöök B., Macellaro A., Sjöstedt A., Norlander L., Early cytokine induction in mouse P388D1 macrophages infected by Coxiella burnetii, Vet. Immunol. Immunopathol. 68 (1999) 159–168. [71] Izzo A.A., Marmion B.P., Variation in interferon-gamma response to Coxiella burnetii antigens with lymphocytes from vaccinated or naturally infected subjects, Clin. Exp. Immunol. 94 (1993) 507–515.
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