Coxiella burnetii: Characteristics, Pathogenesis, Diagnosis microbiologystudy

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

• It is the causative agent of acute and chronic Q fever or Coxiellosis and resembles Ricketssia in terms of morphology but with some genetic and physiological differences.
• C. burnetii belongs to the gammaproteobacteria class that includes other medically important bacteria like Escherichia coli, Salmonella Typhi and Klebsiella pneumoniae.
• It is a coccobacillus bacterium that is resistant to environmental stresses like temperature and acidity.
• C. burnetii exhibits phase variation as a result of antigenic shift. The phase I is highly infectious and occurs in animals, whereas phase II is noninfectious and is observed in cells or embryonated eggs.
• The primary reservoir of C. burnetii is tick which is then transmitted to animals like sheep, goat, wild animals and pets.
• The transmission of bacteria to humans is rare but might occur through aerosol transmission. The ability of C. burnetii to cause infections in humans, along with its environmental stability makes it a potential biological weapon.
• C. burnetii is worldwide in distribution that is a zoonotic pathogenic agent capable of transmitting to vertebrates like humans.
• The isolates of C. burnetii from environmental samples exhibit a variety of phylogenetic homogeneity in the 16S rRNA gene sequencing and DNA-DNA hybridization.
• In the 1920s, the bacteria were isolated from ticks and identified as Rickettsia, which was then renamed as Coxiella burnetti in 1925.
• Coxiella burnetii was named in recognition of the efforts of Cox and Burnett in the identification of the pathogen.
• C. burnetii is also considered a category B bioterrorism agent which is highly infective to humans and livestock.

Classification of Coxiella burnetii

• Coxiella has been closely associated with members of Rickettsiae based on the comparisons of their phenotypic characteristics.
• Later, based on DNA sequences and 16S rRNA sequence, C. burnetii was reclassified into the γ subgroup of Proteobacteria, making it a close relative of species like Legionella pneumophila and Wolbachia persica.
• The differentiation of C. burnetii from Rickettsia was primarily based on the ability of C. brunette to cause intracellular growth in a membrane-bound vacuole as Ricketssia species only replicate in the host cytoplasm.
• The historical classification of C. burnetii within the α-1 subgroup of the Proteobacteria in the Rickettsiales order, the Ricketsiaceae family, is based on the intracellular nature, similar staining characteristics and carriage by ticks.
• The classification was then improved to place the bacteria in the domain Bacteria, Phylum Proteobacteria, Order Legionellales and family Coxiellaceae.
• The Coxiellaceae family further includes other bacteria like Rickettsiella grylii, which is an intracellular parasite of crickets.
• The distinction of C. burnetii from Rickettsia on a phenotypic level by displayed pronounced extracellular stability and resistance to chemical and physical disruption.
• The genotypic differentiation of C. burnetii divides the species into six genomic groups (I to VI).
• The following is the taxonomical classification of C. burnetii;

Domain	Bacteria
Phylum	Proteobacteria
Class	Gammaproteobacteria
Order	Legionellales
Family	Coxiellaceae
Genus	Coxiella
Species	C. burnetii

Habitat of Coxiella burnetii

• Coxiella burnetii is worldwide in distribution and occurs in a different natural environment due to its high degree of stability under unfavourable conditions.
• The transmission and distribution of C. burnetii in a different environment and to different animals and humans is through aerosol transmission.
• The host range is diverse, ranging from arthropods, fish, birds and a variety of vertebrates and mammals.
• The organism has a worldwide distribution with the exception of Antarctic regions and New Zealand.
• The occurrence of C. burnetii occurs in both wild and domestic animals, but the disease is seen only in sheep and goats.
• Infected animals shed the organism in faeces, urine, milk and birth products, but some isolates can be obtained from the lung, liver, spleen and blood samples.
• The most important vector for the transmission and distribution of C. burnetii is tick as it is important in natural infection.
• The bacteria target the uterus and the mammary glands in female mammals which can persist for a longer period of time.
• The ability of C. burnetii to survive in extreme environmental conditions enables the occurrence of bacteria in different habitats.
• C. burnetii is remarkably resistant to desiccation and ultraviolet radiation and can persist in contaminated soils.
• The contaminated aerosol is the primary route of transmission of C. burnetii in humans, whereas a low intraperitoneal infectious dose of C. burnetii in guinea pig models.
•  In ticks and other insects, the bacteria replicate in the midgut or stomach as an obligate intracellular bacterium.

Morphology of Coxiella burnetii

• The cells of C. burnetii are small pleomorphic cells ranging in size from 0.5-0.8 µm to 1.2-3.0 µm. The size of the cell varies greatly as the shape is coccobacillus.
• It is an obligate intracellular organism that replicates within the phagolysosome of host cells. The bacteria combine adaptation to the acid environment of the phagolysosome with a developmental cycle consisting of transverse binary fission and sporogenesis.
• C. burnetii is a spore-like particle forming bacterium where the size of the cell doubles in size with the formation of spore. The cells are not encapsulated and non-motile with no flagella. 
• The spores produced by C. burnetii cells are developed as a result of environmental resistance.
• The cells display a prototypic Gram-negative cell wall composed of peptidoglycan with N-acetylmuramic acid and N-acetylglucosamine, D-alanine, D-glutamic acids and meso-diaminopimelic acid.
• The cells also have an outer lipopolysaccharide membrane where the chemical composition of the outer membrane results in antigenic variation.
• Two antigenic forms of C. burnetii exist depending on the complexity of the lipopolysaccharide layer.
• Phase I C. burnetii replicates in the cells of immunocompetent hosts, lipopolysaccharides are synthesized in its full length along with the cell wall antigens.
• Phase I is extremely contagious to humans as the lipopolysaccharide acts as a virulence factor that induces an immunogenic reaction.
• Phase II cells do not have the full complement of properties required for survival in macrophages. These cells appear after repeated cultivation in the yolk sac of the chicken embryo.

Cultural Characteristics of Coxiella burnetii

• As C. burnetii is an obligate intracellular bacterial pathogen which grows exclusively in the acidified lysosome-like vacuole, the growth of the bacteria cannot be obtained on a nutrient medium.
• The growth and isolation of Coxiella burnetii can be achieved on a cell-free medium like yolk sac of an embryonated chicken embryo.
• The chicken embryo acts as a good medium for the isolation of the bacteria. The eggs used for the inoculation of the bacteria are usually 5-7 days old.
• The space on the top of the air sac is sterilized to inoculate the embryonated egg with the prepared inoculum.
• The inoculum for such preparation is prepared on brain-heart infusion broth or sucrose phosphate glutamate solution.
• Now cell cultures have been developed for the rapid isolation and identification of C. burnetii from infected animals.
• The shell vial culture method allows the concentration of moderate volume of inoculums over a monolayer of highly susceptible cells.
• Another method of isolation of C. burnetii is via plaque formation, which is performed on monolayers of chicken embryo fibroblasts.
• The handling of C. burnetii samples for the isolation and maintenance of the bacteria requires a Biosafety Level 3 facility as the bacteria posses a substantial risk to the laboratory personnel.
• The bacteria can be maintained in liquid nitrogen but more commonly at -80°C. 
• The differentiation of Coxiella species from Rickettsia can be made from the growth of the bacteria on the cell cultures. Rickettsia lacks large particles present intracellularly that later condense to reform the smaller forms. 

Biochemical Characteristics of Coxiella burnetii

The biochemical characteristics of C. burnetii are not known as the bacteria is grown as intracellular bacteria on cell lines and tissue cultures.

Pathogenesis of Coxiella burnetii

• Human infection usually follows inhalation of aerosols containing C. burnetii.
• It is estimated that only between 1 and 10 bacteria are necessary to cause infection.
• C. burnetii has also been known to enter the body via other mucous membranes, abrasions, and the gastrointestinal tract through consumption of milk from infected animals.
• C. burnetii exists in two antigenic forms called phase I and phase II.
• Phase I is the virulent form that is found in humans with Q fever and infected vertebrate animals, and it is the infectious form, whereas Phase II is the avirulent form.
• Entry into the lungs results in infection of the alveolar macrophages.
• C. burnetii escapes intracellular killing in macrophages by:
  • Inhibiting the final phagosome maturation step (cathepsin fusion)
  • Resistant to the acidic environment of phagolysosome by producing superoxide dismutase.
• The normal progression after phagocytosis of most organisms is fusion of the phagosome with a series of endosomes (intracellular vesicles), resulting in a drop in intracellular pH, followed by fusion with lysosomes containing hydrolytic enzymes and resultant bacterial death which occurs with C. burnetii if phase II organisms are ingested; however, phase I Coxiella is able to arrest this process before lysosomal fusion.
• In addition, the organisms require acid pH for their metabolic activities, which, in turn, protects them from the killing activities of most antibiotics.
• Coxiella is able to regulate the cell signaling pathways in its phagocytic home so that cell death is delayed.
• The ability of C. burnetii to cause either acute or chronic disease is determined in part by the organism’s ability to survive intracellularly.
• In acute cases, in the presence of interferon-γ, phagosome–lysosome fusion occurs, leading to bacterial death; however, in chronic infections interleukin-10 is overproduced by the host cell, which interferes with fusion and allows intracellular survival of C. burnetii.
• Infection with C. bumetii induces autoantibodies, particularly to cardiac and smooth muscles.
• Chronic form leads to disseminated cases affecting various organs with pathological condition.

Clinical Manifestations of Coxiella burnetii

Query fever (Q fever)

• The majority of individuals exposed to C. burnetii have an asymptomatic infection, and most symptomatic infections are mild, presenting with nonspecific flulike symptoms with an abrupt onset, high-grade fever, fatigue, headache, and myalgias.
• The patient may also suffer pneumonitis, hepatic and bone marrow granulomata, and meningoencephalitis.
• Hepatitis is usually asymptomatic or presents with fever and increase in serum transaminases.
• Most cases of pneumonia are mild, with a nonproductive cough, fever, and nonspecific findings on chest radiograph.
• Acute pneumonia and hepatitis are associated with antibodies to phase II antigens.
• Chronic infections can develop, with the organism persisting in cardiac valves and possibly other foci.
• Chronic Q fever (symptoms lasting more than 6 months) can develop months to years after the initial exposure and occurs almost exclusively in patients with predisposing conditions, such as underlying valvular heart disease or immunosuppression.
• Fever is usually absent or of low grade.
• Reactivation of latent infection may occur during pregnancy, and the organism is shed with the placenta or abortus.

Laboratory Diagnosis of Coxiella burnetii

Specimen: Blood, tissues from cardiac valve

Culture

• Culture can be performed in tissue culture cells using human embryonic lung fibroblast cell lines, and recently in a cell-free medium; however, culture is rarely performed except in research laboratories licensed to work with these highly contagious organisms.

Serology

• Serology is the most commonly used diagnostic test.
• A variety of methods are used to measure antibody production: the microagglutination tests, indirect immunofluorescence antibody (IFA) test, and enzyme-linked immunosorbent assay (ELISA).
• IFA is the test of choice, although ELISA is used in many laboratories and appears to be as sensitive.
• In chronic infections, the antibodies to phase I antigens are elevated whereas in acute Q fever, immunoglobulins IgM and IgG antibodies are developed primarily against phase II antigens.
• Complement fixation test can also be done detecting IgG antibodies to phase II antigens.
• A diagnosis of chronic Q fever is confirmed by the demonstration of antibodies against both phase I and II antigens, with the titers to the phase I antigen typically higher.

Molecular Methods

• PCR amplification has been used to detect C. burnetii DNA in clinical samples from acute and chronic Q fever patients.
• Strains of C. burnetii differ in their plasmids which they carry.
• QpH1 plasmids are found in acute Q fever isolates; whereas QpRS plasmids are found on the strains isolated from endocarditis patients.

Treatment of Coxiella burnetii

• Most infections resolve without antibiotic treatment, but administration of doxycycline reduces the duration of fever in the acute infection and is definitely recommended in cases of chronic infection.
• Fluoroquinolones (e.g., ofloxacin, pefloxacin) have been used as an alternative to doxycycline but are contraindicated in children and pregnant women.
• The newer macrolides have also been shown to be effective in the treatment of acute pneumonia.
• Chronic Q fever requires prolonged treatment for 18 months or longer with a combination of doxycycline and hydroxychloroquine.
• In Q-fever endocarditis, long-term administration of a combination of two drugs, combination therapy among doxycycline, ciprofloxacin and rifampicin has been suggested to prevent relapse.

Prevention and Control of Coxiella burnetii

• The presently recommended conditions of “high-temperature, short-time” pasteurization at 71.5°C for 15 seconds are adequate to destroy viable Coxiella species.
• Exposure can be reduced by construction of separate facilities for animal parturition, destruction of suspect placental membranes,heat treatment of milk and efforts to reduce the tick population.
• Occupationally exposed persons may reduce their risk of infection with burnetii by wearing respirators that prevent aerosol infections.
• Inactivated whole-cell vaccine (Q-Vax) and partially purified antigen vaccines for Q fever have been developed, and the vaccines prepared from phase I organisms have been shown to provide the best protection and is recommended for occupationally exposed workers.
• Good animal husbandry practices should be followed such as proper disposal of animal excreta and aborted materials, isolation of aborting animals for 14 days.

References

1. Rickettsia, orientia, ehrlichia, anaplasma and coxiella. (n.d.). Retrieved from https://www.researchgate.net/publication/309496161_Rickettsia_orientia_ehrlichia_anaplasma_and_coxiella
2. Heinzen, R.A., Samuel, J.E. (2006). The Genus Coxiella . In: Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, KH., Stackebrandt, E. (eds) The Prokaryotes. Springer, New York, NY. https://doi.org/10.1007/0-387-30745-1_21
3. Neupane K, Kaswan D. Coxiella burnetii Infection. [Updated 2023 May 22]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557893/
4. Roest, H. I. J., Bossers, A., van Zijderveld, F. G., & Rebel, J. M. L. (2013). Clinical microbiology of Coxiella burnetii and relevant aspects for the diagnosis and control of the zoonotic disease Q fever. Veterinary Quarterly, 33(3), 148–160. https://doi.org/10.1080/01652176.2013.843809
5. Gürtler, L., Bauerfeind, U., Blümel, J., Burger, R., Drosten, C., Gröner, A., Heiden, M., Hildebrandt, M., Jansen, B., Offergeld, R., Pauli, G., Seitz, R., Schlenkrich, U., Schottstedt, V., Strobel, J., & Willkommen, H. (2014). Coxiella burnetii – Pathogenic Agent of Q (Query) Fever. Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie, 41(1), 60–72. https://doi.org/10.1159/000357107
6. Bauer, B. U., Knittler, M. R., Andrack, J., Berens, C., Campe, A., Christiansen, B., . . . Lührmann, A. (2023). Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research. International Journal of Medical Microbiology, 313(6), 151590. https://doi.org/10.1016/j.ijmm.2023.151590
7. Omsland A, Cockrell DC, Howe D, et al. Host cell-free growth of the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A. 2009;106(11):4430-4434. doi:10.1073/pnas.0812074106
8. Beard, P. D., Spalatin, J., & Hanson, R. P. (1970). Strain Identification of Newcastle Disease Virus in Tissue Culture. Avian Diseases, 14(4), 636–645. https://doi.org/10.2307/1588635
9. Clinical and laboratory diagnosis for Q fever. (2024, May 15). Retrieved from https://www.cdc.gov/q-fever/hcp/diagnosis-testing/index.html
10. Moodie, C. E., Thompson, H. A., Meltzer, M. I., & Swerdlow, D. L. (2008). Prophylaxis after Exposure to Coxiella burnetii. Emerging Infectious Diseases, 14(10), 1558-1566. https://doi.org/10.3201/eid1410.080576.
11. Dragan, A. L., & Voth, D. E. (2020). Coxiella burnetii: international pathogen of mystery. Microbes and infection, 22(3), 100–110. https://doi.org/10.1016/j.micinf.2019.09.001