Coxiella burnetii: Structure, Properties and Treatment
▶The History of Discovery
For a long time Q fever disease was regarded as rickettsiosis. The first description of febrile disorder that regularly occurred among abattoirs was made in 1937 by E.H. Derrick in Queensland, Australia. Derrick termed it Q fever (short for “query fever”). He was not successful in isolation of its putative causative agent and supposed it to be an unknown virus. Then M. Burnet and M. Freeman reproduced the disease in animals and detected the infectious agent in the vacuoles of infected cells. Similar work was produced by G. Davis and H.R. Cox in the USA, who isolated rickettsia-like causative agent from patients with unusual fever or Nine Mile disease.
Further collaborative studies proved these newly discovered diseases to be identical. The agent was finally termed as Coxiella burnetii – a novel genus within Rickettsiacea family. Only latest phylogenetic investigations relied upon 16S rRNA typing demonstrated considerable divergence between coxiellae and rickettsiae. Today C. burnetii is placed into new separate family in the borders of another microbial class lying apart from rickettsiae.
▶Classification of Q Fever Agent
Q fever agent pertains to the order Legionellales, family Coxiellaciae, genus Coxiella, and species C. burnetii.
▶Structure and Properties of Coxiellae
Coxiellae, similar to rickettsiae, are 0.3 by 0.5-1 μm small size gram-negative bacteria with obligate intracellular parasitism. They are poorly stained with Gram method but can be readily detected by Gimenez stain as well as by Zdrodovsky method. Two main morphological types of bacteria arise in the course of C. burnetii infection. “Small-cell” variant is an extracellular resistant bacterial form with slow metabolism that is capable of invading mammalian phagocytes.
Further it turns into active intracellular “large-cell” form within host phagolysosomes. Both types can multiply by binary fission. In unfavorable conditions (e.g., within phagolysosome) large-cell variants can undergo further transformation into spore-like microbial bodies. The latter are the special bacterial forms resistant to external impacts. Finally, spore-like bodies transform back into small-cell microbials, which spread outside via exocytosis or after lysis of the infected cell. Bacterial genome carries nucleoid and a number of plasmids.
Coxiellae can’t grow in artificial nutrient media. They may be cultured in various cell lines (e.g., human embryo fibroblasts), embryonated eggs and in susceptible animals (guinea pigs, mice, etc). In cell lines the bacteria are detected in 5-7 days after primary inoculation. During persistent infection bacteria show slow propagation with doubling time of about 20 h.
- Biochemical properties
Coxiellae are more biochemically active than rickettsiae; bacteria use their own methabolic pathways for ATP and protein synthesis.
- Antigenic structure
Antigenic composition of coxiellae depends on phase variation of cellular lipopolysaccharide structure that results from the cascade of mutational events. Bacteria with LPS of phase I are isolated from infected animals or humans and regarded as highly infectious bacterial form. They are similar with S forms of other microorganisms. Phase II bacteria appear after multiple passages of coxiellae in cell cultures or embrionated eggs. They are related with rough LPS with altered structure.
- Virulence factors
Toxic and immunosuppressive factors of C. burnetii are not well defined yet. The bacteria produce catalase and superoxide dismutase that inhibit respiratory burst in phagocytes. After degradation microbial cells release endotoxin.
Coxiellae show high resistance in natural environment. The organisms stay viable for about 1 year at 4ºC. Heating at 70-90ºC only partially inactivates bacteria, while 100ºC heating kills them within 10 minutes. They resist desiccation, as well as low and high pH conditions. Disinfectants work slowly against coxiellae; the bacteria can withstand the action of formaldehyde and carbolic acid but show sensitivity to alcohols and ether.
▶Pathogenesis and Clinical Findings in Q fever
Q fever is a zoonotic ubiquitous disease that affects various mammals, birds and arthropods (ticks). Ticks maintain transovarial transmission of coxiellae. The main sources of infection for humans are domestic animals and pets (cattle, goats, sheep, cats, dogs, etc.) The infected animals excrete bacteria with urine and feces. The disease is regarded now as occupational hazard in staff working with domestic animals.
The disease is transmitted predominantly by airborne (aerosol) route after inhalation of dust from contaminated fomites. Fecal-oral transmission is seldom; it occurs mostly after drinking raw milk. Other variants of disease contraction are extremely rare. Incubation period of illness lasts for about 2-3 weeks. Coxiellae can persist only within the phagocytes of the host.
Phase I bacteria enter human phagocytes via membrane integrins. After internalization, they appear within phagolysosomes that results in large vacuole formation. Bacteria are extremely resistant to acidic pH 4.7-5.2 of phagolysosomes thereby maintaining their capability to multiply within phagocytes (incomplete phagocytosis).
Coxiellae can modulate host immune response. They block reactive oxygen species of phagocytes producing catalase and superoxide dismutase. Bacteria depress T cell response and cause T helper lymphopenia partially via induction of suppressive cytokine synthesis by host immune cells. On the other hand, they provoke body inflammation stimulating the synthesis of TNF-α and γ-interferon. The cell-mediated reactions of delayed hypersensitivity are common.
About 60% of disease cases are asymptomatic or may develop mild symptoms. Nevertheless, the rest of cases are severe, especially in immunocompromised patients. Acute Q fever is characterized by sudden onset with high fever, chills and headaches. Two main clinical syndromes (severe atypical pneumonia and granulomatous hepatitis) are usually common depending predominantly on aerosol or foodborne microbial transmission. Myocarditis and meningoencephalitis may rarely occur.
Chronic Q fever is developed in 6 month after primary infection. It is a potentially fatal disease that is manifested by life-threatening endocarditis and inflammatory syndrome. The endocarditis appears to be highly resistant to antimicrobial therapy. Post-infectious immunity is rather stable, cellular reactions play a predominant role in body protection.
▶Laboratory Diagnosis of Q fever
Specimens are collected from patient’s blood and tissue biopsies. Serum is used for serological examination. To determine bacteria in tissue specimens indirect immunofluorescence technique is applied. Serological testing dominates in routine laboratory practice to confirm Q fever diagnosis.
Indirect immunofluorescence and complement fixation test are most widely used. Antibodies against the microbial antigens of both I and II phase are determined. The rise of antibody IgG titer against phase II antigens is characteristic for acute Q fever, while antibodies against the antigens of phase I prevail in chronic disease. Microagglutination test, dot immunoblotting and ELISA are also available.
Cultivation of coxiellae, as well as animal experimental infection, is possible only in specialized laboratories of biosafety level 3 due to high infectivity of Q fever causative agent. The material is inoculated into various cell lines, embryonated eggs and susceptible animals (guinea pigs or mice). Molecular methods, including PCR, are progressively introduced now into laboratory practice to diagnose Q fever.
▶Prophylaxis and Treatment of Q fever
C. burnetii is primarily resistant to beta-lactam and aminoglycoside antibiotics. Therefore, macrolides, doxycycline and fluoroquinolones are the drugs of choice for treatment of acute Q fever. It is much more difficult to cure chronic Q fever endocarditis. Treatment schemes that include combined therapy with doxycycline and fluoroquinolones at least for 1-3 years are introduced into clinical practice.
For specific prophylaxis of Q fever various live, formaldehyde-treated, and chemical vaccines were proposed. They are used to protect the persons with occupation of risk. Nevertheless, these vaccines develop various adverse effects and usually confer the immunity of short duration.