Coxiella burnetii: The Causative Agent of Q Fever - A Comprehensive Guide

Table of Contents

• Introduction to Coxiella burnetii
• Classification of Coxiella burnetii
• Habitat of Coxiella burnetii
• Morphology of Coxiella burnetii
• Cultural Characteristics of Coxiella burnetii
• Biochemical Characteristics of Coxiella burnetii
• Pathogenesis of Coxiella burnetii
• Clinical Manifestations of Coxiella burnetii
• Query fever (Q fever)
• Laboratory Diagnosis of Coxiella burnetii
• Culture
• Serology
• Molecular Methods
• Treatment of Coxiella burnetii
• Prevention and Control of Coxiella burnetii

Introduction to Coxiella burnetii

Coxiella burnetii is a Gram-negative, acidophilic bacterium that resides intracellularly within the phagolysosome of eukaryotic host cells.

• It is the causative agent of both acute and chronic Q fever, also known as Coxiellosis. Morphologically, it resembles Rickettsia, though there are notable genetic and physiological differences between the two.
• This bacterium belongs to the Gammaproteobacteria class, which includes other clinically significant bacteria such as Escherichia coli, Salmonella Typhi, and Klebsiella pneumoniae.
• C. burnetii is a coccobacillus that demonstrates resistance to environmental stresses, including temperature and acidity.
• The bacterium exhibits phase variation due to antigenic shift; Phase I is highly infectious and typically found in animals, while Phase II is noninfectious and occurs in cultured cells or embryonated eggs.
• The primary reservoir for C. burnetii is ticks, which transmit the bacteria to various animals, including sheep, goats, wild animals, and pets.
• Transmission to humans is uncommon, but it may occur through aerosol routes. Its capacity to infect humans and its environmental resilience contribute to its potential as a biological weapon.
• C. burnetii is globally distributed and serves as a zoonotic pathogen capable of infecting vertebrates, including humans.
• Environmental isolates of C. burnetii show a range of phylogenetic homogeneity based on 16S rRNA gene sequencing and DNA-DNA hybridization studies.
• The bacterium was first isolated from ticks in the 1920s and initially classified as Rickettsia before being renamed Coxiella burnetii in 1925, honoring the contributions of Cox and Burnett in identifying the pathogen.
• C. burnetii is classified as a category B bioterrorism agent due to its high infectivity in both humans and livestock.

Classification of Coxiella burnetii

• Coxiella has historically been linked to members of the Rickettsiae based on their phenotypic traits. However, advances in DNA sequencing and analysis of 16S rRNA sequences led to the reclassification of C. burnetii into the γ subgroup of Proteobacteria, aligning it more closely with species such as Legionella pneumophila and Wolbachia persica.
• The primary distinction between C. burnetii and Rickettsia lies in C. burnetii's ability to grow intracellularly within a membrane-bound vacuole, while Rickettsia species replicate solely in the host cytoplasm.
• Historically, C. burnetii was classified within the α-1 subgroup of Proteobacteria in the Rickettsiales order and the Rickettsiaceae family due to its intracellular nature, similar staining properties, and its association with ticks.
• This classification was refined, placing C. burnetii in the domain Bacteria, phylum Proteobacteria, order Legionellales, and family Coxiellaceae.
• The Coxiellaceae family also includes other bacteria, such as Rickettsiella grylii, an intracellular parasite of crickets.
• Distinguishing C. burnetii from Rickettsia on a phenotypic level is supported by its notable extracellular stability and resistance to chemical and physical disruptions.
• Genotypically, C. burnetii is divided into six genomic groups, designated I to VI.

The following is the taxonomical classification of C. burnetii:

Habitat of Coxiella burnetii

• Coxiella burnetii has a global distribution and thrives in various natural environments due to its remarkable stability under adverse conditions.
• The transmission and spread of C. burnetii occur primarily through aerosol routes, affecting different animals and humans in diverse environments.
• Its host range is extensive, encompassing arthropods, fish, birds, and a wide array of vertebrates and mammals.
• The bacterium is found worldwide, except in Antarctic regions and New Zealand.
• While C. burnetii can be present in both wild and domestic animals, the disease predominantly manifests in sheep and goats.
• Infected animals shed the bacteria through feces, urine, milk, and birthing products; isolates can also be retrieved from the lungs, liver, spleen, and blood samples.
• Ticks are the most significant vector for the transmission and spread of C. burnetii, playing a crucial role in natural infections.
• The bacteria primarily target the uterus and mammary glands in female mammals, where they can persist for extended periods.
• C. burnetii’s ability to endure extreme environmental conditions allows it to inhabit a variety of ecosystems.
• The bacterium shows exceptional resistance to desiccation and ultraviolet radiation, enabling it to survive in contaminated soils.
• The primary mode of transmission to humans is through contaminated aerosols, while a low intraperitoneal infectious dose has been observed in guinea pig models.
• In ticks and other insects, C. burnetii replicates in the midgut or stomach as an obligate intracellular bacterium.

Morphology of Coxiella burnetii

• Coxiella burnetii consists of small, pleomorphic cells that range in size from 0.5-0.8 µm to 1.2-3.0 µm, exhibiting a coccobacillus shape.
• It is an obligate intracellular organism that replicates within the phagolysosome of host cells, adapting to the acidic environment of the phagolysosome and employing a developmental cycle that includes transverse binary fission and sporogenesis.
• C. burnetii produces spore-like particles, with the size of the cells doubling during spore formation. The cells are non-capsulated, non-motile, and lack flagella.
• The spores generated by C. burnetii are a response to environmental stressors, enhancing the organism's resilience.
• The cells possess a typical Gram-negative cell wall made up of peptidoglycan, which includes components such as N-acetylmuramic acid, N-acetylglucosamine, D-alanine, D-glutamic acid, and meso-diaminopimelic acid.
• Additionally, they feature an outer membrane rich in lipopolysaccharides, contributing to antigenic variation.
• C. burnetii exists in two antigenic forms, which depend on the complexity of the lipopolysaccharide layer.
• Phase I cells replicate in immunocompetent hosts, where lipopolysaccharides are synthesized in their complete form, along with cell wall antigens. This phase is highly contagious to humans because the lipopolysaccharides act as a virulence factor that triggers an immunogenic response.
• Phase II cells lack the full array of properties necessary for survival within macrophages and appear after repeated cultivation in the yolk sac of chicken embryos.

Cultural Characteristics of Coxiella burnetii

• Coxiella burnetii is an obligate intracellular bacterial pathogen that exclusively grows within acidified lysosome-like vacuoles, making it impossible to culture on standard nutrient media.
• Isolation and growth of C. burnetii can be accomplished using cell-free media, such as the yolk sac of an embryonated chicken embryo, which serves as an effective medium for this purpose.
• Typically, the chicken embryos used for inoculation are 5-7 days old. The area above the air sac is sterilized before inoculating the embryonated egg with the prepared inoculum.
• The inoculum is often prepared using brain-heart infusion broth or a sucrose phosphate glutamate solution.
• Recently developed cell cultures facilitate the rapid isolation and identification of C. burnetii from infected animals.
• The shell vial culture method enables the concentration of moderate volumes of inoculum over a monolayer of highly susceptible cells.
• Another isolation technique involves plaque formation on monolayers of chicken embryo fibroblasts.
• Handling samples of C. burnetii for isolation and maintenance requires a Biosafety Level 3 facility due to the significant risk posed to laboratory personnel.
• The bacteria can be stored in liquid nitrogen but are more commonly maintained at -80°C.
• Differentiation between Coxiella species and Rickettsia can be achieved based on their growth patterns in cell cultures. Unlike Rickettsia, C. burnetii exhibits large intracellular particles that condense to form smaller forms.

Biochemical Characteristics of Coxiella burnetii

The biochemical characteristics of Coxiella burnetii remain largely unknown because the bacteria are cultured as intracellular organisms within cell lines and tissue cultures.

Pathogenesis of Coxiella burnetii

• Human infection typically occurs following the inhalation of aerosols containing C. burnetii.
• It is estimated that as few as 1 to 10 bacteria are sufficient to initiate an infection.
• C. burnetii can also enter the body through other mucous membranes, skin abrasions, or the gastrointestinal tract, often via the consumption of milk from infected animals.
• The bacterium exists in two antigenic forms: Phase I and Phase II.
• Phase I is the virulent form associated with Q fever in humans and infected vertebrate animals, while Phase II is considered avirulent.
• Upon entering the lungs, C. burnetii infects alveolar macrophages.
• The bacterium evades intracellular killing within macrophages by:
• Inhibiting the final maturation step of the phagosome (cathepsin fusion).
• Withstanding the acidic environment of the phagolysosome by producing superoxide dismutase.
• Normally, after phagocytosis, most organisms undergo phagosome fusion with various endosomes, resulting in a decrease in intracellular pH followed by fusion with lysosomes containing hydrolytic enzymes, leading to bacterial death. However, if C. burnetii is in the Phase II form, it is susceptible to this process; Phase I C. burnetii, on the other hand, can halt this process prior to lysosomal fusion.
• Additionally, the bacteria require an acidic pH for their metabolic functions, which protects them from the action of many antibiotics.
• C. burnetii can manipulate cell signaling pathways in its phagocytic environment to delay cell death.
• The bacterium's ability to induce either acute or chronic disease is partly dependent on its capacity to survive intracellularly.
• In acute cases, the presence of interferon-γ promotes phagosome-lysosome fusion, resulting in bacterial death. Conversely, chronic infections see an overproduction of interleukin-10 by host cells, which inhibits fusion and allows C. burnetii to persist intracellularly.
• Infection with C. burnetii can lead to the production of autoantibodies, particularly against cardiac and smooth muscle tissues.
• The chronic form of the infection can result in disseminated cases affecting multiple organs and leading to various pathological conditions.

Clinical Manifestations of Coxiella burnetii

Query fever (Q fever)

• Most individuals exposed to C. burnetii experience asymptomatic infections, while symptomatic cases are generally mild, presenting with nonspecific flu-like symptoms that have an abrupt onset, including high-grade fever, fatigue, headache, and myalgia.
• Patients may also experience complications such as pneumonitis, hepatic and bone marrow granulomatosis, and meningoencephalitis.
• Hepatitis is typically asymptomatic or may present with fever and elevated serum transaminases.
• Most pneumonia cases are mild, characterized by a nonproductive cough, fever, and nonspecific findings on chest radiographs.
• Acute pneumonia and hepatitis are linked to the presence of antibodies against phase II antigens.
• Chronic infections can develop, with the organism persisting in cardiac valves and possibly other locations.
• Chronic Q fever, defined as symptoms lasting longer than six months, may emerge months to years after initial exposure and almost exclusively affects individuals with predisposing factors, such as underlying valvular heart disease or immunosuppression.
• In chronic cases, fever is typically absent or only low grade.
• Reactivation of latent infections can occur during pregnancy, leading to the shedding of the organism with the placenta or during abortion.

Laboratory Diagnosis of Coxiella burnetii

Specimens: 

• Blood and tissue samples from cardiac valves.

Culture

• Culture can be conducted using tissue culture cells, such as human embryonic lung fibroblast cell lines, and more recently in cell-free media. However, culturing C. burnetii is infrequent outside of licensed research laboratories due to the organism's highly contagious nature.

Serology

• Serological testing is the most widely used diagnostic method. Various techniques measure antibody production, including microagglutination tests, indirect immunofluorescence antibody (IFA) tests, and enzyme-linked immunosorbent assays (ELISA). 
• The IFA test is preferred, although ELISA is also common and appears to have comparable sensitivity. 
• In chronic infections, antibodies against phase I antigens are elevated, while acute Q fever is characterized by the development of immunoglobulins IgM and IgG primarily against phase II antigens. 
• A complement fixation test can also detect IgG antibodies against phase II antigens. 
• Chronic Q fever is confirmed by demonstrating antibodies against both phase I and II antigens, with titers for phase I typically being higher.

Molecular Methods

• PCR amplification has been utilized to detect C. burnetii DNA in clinical samples from patients with both acute and chronic Q fever. 
• Strains of C. burnetii vary in their plasmids: QpH1 plasmids are associated with acute Q fever isolates, while QpRS plasmids are linked to strains isolated from endocarditis patients.

Treatment of Coxiella burnetii

• Most C. burnetii infections resolve without the need for antibiotic treatment. However, administering doxycycline is recommended to reduce the duration of fever in acute infections and is essential in cases of chronic infection.
• Fluoroquinolones (such as ofloxacin and pefloxacin) can serve as alternatives to doxycycline but are contraindicated for use in children and pregnant women.
• Newer macrolides have demonstrated effectiveness in treating acute pneumonia caused by C. burnetii.
• Chronic Q fever requires prolonged treatment lasting 18 months or more, typically involving a combination of doxycycline and hydroxychloroquine.
• For Q fever endocarditis, long-term therapy with a combination of medications, including doxycycline, ciprofloxacin, and rifampicin, has been recommended to prevent relapse.

Prevention and Control of Coxiella burnetii

• The currently recommended "high-temperature, short-time" pasteurization method, which involves heating at 71.5°C for 15 seconds, is effective in eliminating viable Coxiella species.
• Reducing exposure can be achieved by constructing separate facilities for animal birthing, disposing of suspected contaminated placental membranes, heat-treating milk, and implementing measures to decrease the tick population.
• Individuals in occupations with potential exposure can lower their risk of infection by wearing respirators designed to prevent aerosol transmission.
• An inactivated whole-cell vaccine (Q-Vax) and partially purified antigen vaccines for Q fever have been developed. Vaccines derived from phase I organisms offer the highest level of protection and are recommended for workers at risk.
• Implementing good animal husbandry practices is essential, including the proper disposal of animal waste and aborted materials and isolating aborting animals for at least 14 days.