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Table of Contents:
Taxonomy Information
  1. Species:
    1. Coxiella burnetii (Website 6):
      1. GenBank Taxonomy No.: 777
      2. Description: Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular gram-negative bacterium classified in the Rickettsiaceae family and belonging to the gamma subdivision of Proteobacteria(Maurin and Raoult, 1999). Although Coxiella was historically considered "Rickettsia-like," 16S rRNA gene sequence analysis and genome analysis (based on shared proteins across genomes and phylogenetic analysis of a set of 20 highly conserved proteins) indicate that it is a gamma proteobacteria (order Legionellales) and thus is distant from the gamma proteobacterial Rickettsia group. Coxiella is also distant from any other lineage within the subgroup, its closest relationship is with Legionella pneumophila, a facultative intracellular human pathogen, and Rickettsiella grylli, an intracellular arthropod pathogen(Seshadri et al., 2003). C. burnetii is very resistant to killing in nature and is further able to survive in the acidic environment of phagolysosomes(Maurin and Raoult, 1999).
      3. Variant(s):
        • Coxiella burnetii Group I (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: DNA from 38 Coxiella burnetii isolates was examined by restriction fragment length polymorphism (RFLP) analysis, resulting in the description of six genomic groups (I to VI). Genomic Group I is associated with animal, tick, or acute Q fever isolates (referred to as acute strains) and contains the plasmid QpH1(Maurin and Raoult, 1999).
        • Coxiella burnetii Group II (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: DNA from 38 Coxiella burnetii isolates was examined by restriction fragment length polymorphism (RFLP) analysis, resulting in the description of six genomic groups (I to VI). Genomic Group II is associated with animal, tick, or acute Q fever isolates (referred to as acute strains) and contains the plasmid QpH1(Maurin and Raoult, 1999).
        • Coxiella burnetii Group III (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: DNA from 38 Coxiella burnetii isolates was examined by restriction fragment length polymorphism (RFLP) analysis, resulting in the description of six genomic groups (I to VI). Genomic Group III is associated with animal, tick, or acute Q fever isolates (referred to as acute strains) and contains the plasmid QpH1(Maurin and Raoult, 1999).
        • Coxiella burnetii Group IV (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: DNA from 38 Coxiella burnetii isolates was examined by restriction fragment length polymorphism (RFLP) analysis, resulting in the description of six genomic groups (I to VI). Genomic Group IV is associated with human Q fever endocarditis isolates (referred to as chronic strains) and contains the plasmid QpRS(Maurin and Raoult, 1999).
        • Coxiella burnetii Group V (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: DNA from 38 Coxiella burnetii isolates was examined by restriction fragment length polymorphism (RFLP) analysis, resulting in the description of six genomic groups (I to VI). Genomic Group V is associated with human Q fever endocarditis isolates (referred to as chronic strains) and is plasmidless. However, Group V isolates were found to contain chromosome-integrated DNA sequences with homology to the QpRS plasmid(Maurin and Raoult, 1999).
        • Coxiella burnetii Group VI (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: DNA from 38 Coxiella burnetii isolates was examined by restriction fragment length polymorphism (RFLP) analysis, resulting in the description of six genomic groups (I to VI). Genomic Group VI isolates (obtained from feral rodents) are of unknown pathogenicity and contain the plasmid QpDG(Maurin and Raoult, 1999).
        • Coxiella burnetii Nine Mile Strain (Maurin and Raoult, 1999):
        • Coxiella burnetii French Strain (Maurin and Raoult, 1999):
        • Coxiella burnetii Small-Cell Variants (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: Small-cell variants (SCVs) are metabolically inactive and resistant to osmotic pressure and correspond to the extracellular form of Coxiella burnetti. SCVs attach to the eukaryotic cell membrane to enter phagocytic cells. After phagolysosomal fusion, acid activation of the metabolism of SCVs may lead to the formation of large-cell variants (LCVs). SCVs have a typical eubacterial gram-negative cell wall with two membranes separated by a periplasmic space. A dense material fills the periplasmic space in SCVs and corresponds to proteins and peptidoglycan. This material may explain the increased resistance of SCVs to environmental conditions(Maurin and Raoult, 1999).
        • Coxiella burnetii Small-dense-cell Variants (Heinzen et al., 1999):
        • Coxiella burnetii Large-Cell Variants (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: Large-cell variants (LCVs) correspond to the metabolically active intracellular form of Coxiella burnetii. A sporogenic differentiation has been characterized in LCVs, leading to the formation of resistant, spore-like forms of bacteria. The endogenous spore-like forms undergo further development to become the metabolically inactive small-cell variants (SCVs), which are then released from the infected host cell either by cell lysis or possibly by exocytosis(Maurin and Raoult, 1999).
        • Coxiella burnetii Phase I (Maurin and Raoult, 1999):
          • Parents: Coxiella burnetii
          • Description: Coxiella burnetii displays antigenic variations similar to the smooth-rough variation in the family Enterobacteriaceae. Phase variation is related mainly to mutational variation in the lipopolysaccharide (LPS). Phase I is the highly infectious, natural phase of Coxiella burnetii corresponding to smooth lipopolysaccharide (LPS). Phase I is found in infected animals, arthropods, or humans(Maurin and Raoult, 1999). This antigenic variation is extremely valuable for the serological differentiation between acute and chronic Q fever as the presence of anti-phase I antibodies is indicative of chronic Q fever(Maurin and Raoult, 1999, Fournier et al., 1998).
        • Coxiella burnetii Phase II (Maurin and Raoult, 1999):
          • Common Name: Phase II
          • Parents: Coxiella burnetii
          • Description: Coxiella burnetii displays antigenic variations similar to the smooth-rough variation in the family Enterobacteriaceae. Phase variation is related mainly to mutational variation in the lipopolysaccharide (LPS). Phase II is the "not very infectious" phase of Coxiella burnetii obtained only after serial passages in cultures. It corresponds to rough lipopolysaccharide (LPS). Compared to phase I, phase II displays a truncated LPS and lacks some protein cell surface determinants(Maurin and Raoult, 1999). This antigenic variation is extremely valuable for the serological differentiation between acute and chronic Q fever as the presence of anti-phase II antibodies is indicative of acute Q fever(Maurin and Raoult, 1999, Fournier et al., 1998).
        • Coxiella burnetii large cell variants (Madariaga et al., 2003):
          • Common Name: Coxiella burnetii large cell variants
          • Parents: Coxiella burnetii
          • Description: Electron microscopy suggests that coxiella has two morphological forms (large and small), a phenomenon different from the phase variation phenomenon and the developmental stages of the organism. Coxiella is ingested as a small variant form by the host cell. Fusion of the primary lysosome and the phagosome containing the organism occurs. The acid pH of the phagolysosome activates the enzymes of C. burnetii and leads to development of the large cell variant, which in turn may produce highly resistant spores. Six strain types have been described: Hamilton, Bacca, Rasche, Biothere, Corazon and Dod(Madariaga et al., 2003).
Lifecycle Information
    1. Stage Information:
      1. Vegetative Cell:
        • Size: Small, 0.2 to 0.4 microns wide, 0.4 to 1 microns long (Maurin and Raoult, 1999). The small-cell variants: 0.2 to 0.5 microns; Length can exceed 1.0 micron for large-cell variants
        • Shape: Pleomorphic rod. Rod-like, compact. Large-cell variants are more pleomorphic than the small-cell variants.
Genome Summary
  1. Genome of Coxiella burnetii Nine Mile Strain
    1. Description: The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method(Seshadri et al., 2003). However, the genome size is highly variable among different C. burnetii strains, ranging from 1.5 to 2.4 Mb(Maurin and Raoult, 1999). Although previously suspected to be linear, genome sequencing and analysis suggest a circular topology for the chromosome. The genome is predicted to encode 2,134 CDSs, of which 719 (33.7%) are hypothetical, i.e., have no significant matches to other sequenced genes, which is a high proportion given the large number of gamma proteobacterial genome sequences currently available. Genome analysis identified 83 "pseudogenes" (genes disrupted by one or more authentic frameshifts and/or point mutations or, in some cases, truncations) suggesting that some genome reduction is underway(Seshadri et al., 2003). Genomic sequence data indicate that 21 genes encoding products that are similar to components of the Legionella pneumophila Dot/Icm type IV secretion system are located on a contiguous 35 kb region of the Coxiella chromosome(Zamboni et al., 2003).
    2. Chromosome(Website 8)
      1. GenBank Accession Number: NC_002971
      2. Size: 2.1 Mb(Willems et al., 1998, Maurin and Raoult, 1999).
      3. Description: The chromosome shape is probably linear(Willems et al., 1998, Maurin and Raoult, 1999).
    3. Plasmid QpH1(Website 9)
      1. GenBank Accession Number: NC_002118
      2. Size: 37 kb(Website 9).
      3. Description: Plasmid QpH1 is found in genomic groups I, II, and III and is thus associated with acute Coxiella burnetii strains(Maurin and Raoult, 1999).
    4. Plasmid QpRS
      1. Size: 39 kb(Maurin and Raoult, 1999).
      2. Description: Plasmid QpRS is a 39-kb plasmid found in genomic group IV. This plasmid was found in Coxiella burnetii Priscilla, obtained from an aborted goat fetus. The QpRS plasmid was also found in human Q fever endocarditis isolates(Maurin and Raoult, 1999).
    5. Plasmid QpDG
      1. Size: 42 kb(Maurin and Raoult, 1999).
      2. Description: Plasmid QpDG is a 42 kb plasmid found in genomic group VI isolates (obtained from feral rodents)(Maurin and Raoult, 1999).
    6. Plasmid QpDV(Website 10)
      1. GenBank Accession Number: NC_002131
      2. Size: 33 kb(Maurin and Raoult, 1999).
      3. Description: Plasmid QpDV is a 33-kb plasmid that has been found in a French strain isolated from an endocarditis patient(Maurin and Raoult, 1999).
    7. Plasmid pQpH1(Website 18)
      1. GenBank Accession Number: NC_004704
      2. Size: 37 kb(Website 18).
      3. Description: Plasmid pQpH1 is found in Coxiella burnetii strain RSA 493(Website 18).
Biosafety Information
  1. Biosafety information for Coxiella burnetii
    1. Level: Depending on the work being done, practices associated with biosafety level 2 or 3 are recommended (C. burnetii: MSDS; Rickettsial agents - Coxiella).
    2. Precautions: Biosafety level 2 practices and containment for nonpropagative laboratory procedures including serological examinations and staining of impression smears. Biosafety level 3 practices and containment for activities involving the inoculation, incubation, and harvesting of embryonated eggs or tissue cultures, the necropsy of infected animals, and the manipulation of infected tissues(Website 1, Website 13, Bernard et al., 1982, Spinelli et al., 1981). Protective clothing including laboratory coats, gloves, and gown (tight wrists and fastened back) should always be worn when working with this organism. Masks may also be used(Website 1). Since infected guinea pigs and other rodents may shed the organisms in urine or feces, experimentally infected rodents should be maintained under Animal Biosafety Level 3. Recommended precautions for facilities using sheep as experimental animals are described in Bernard et al. (1982) and Spinelli et al. (1981). An investigational new Phase I Q fever vaccine (IND) is available from the Special Immunizations Program, U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), Fort Detrick, Maryland. The use of this vaccine should be limited to those at high risk of exposure and who have no demonstrated sensitivity to Q fever antigen. Individuals with valvular heart disease should not work with Coxiella burnetii(Website 13, Bernard et al., 1982, Spinelli et al., 1981).
    3. Disposal: Decontaminate all wastes before disposal: incineration (animal wastes), steam sterilization(Website 1).
Culturing Information
  1. Culturing Overview :
    1. Description: Since Coxiella burnetii is a strict intracellular bacterium, culturing is not possible in axenic medium. Although it has been successfully isolated in guinea pigs, mice, and embryonated eggs, such techniques have been abandoned because they are more hazardous than in vitro cell cultures(Maurin and Raoult, 1999). In vitro, C. burnetii replicates within a wide variety of epithelial, fibroblast, and macrophage-like cell lines(Heinzen et al., 1999). including; human embryonic lung fibroblasts (HEL cells), which are highly susceptible to C. burnetii infection,(Maurin and Raoult, 1999, Raoult et al., 1990). murine macrophage-like cell lines P388D1, J774, and PU-5-IR;(Maurin and Raoult, 1999, Baca et al., 1981). mouse L929 fibroblasts;(Capo et al., 1999). and Buffalo green monkey kidney cells (BGM cell line)(Macellaro et al., 1998).
  2. Culture of Coxiella burnetii in HEL cells :
    1. Description: A system for isolation and routine in vitro culture of Coxiella burnetii in human embryonic lung fibroblasts (HEL cells) was developed by Raoult et al(Maurin and Raoult, 1999, Raoult et al., 1990). HEL cell monolayers were grown in a shell vial until confluent and a sample containing Coxiella burnetii was added to it. To increase the attachment and penetration of the bacteria into the cells, the vial was centrifuged at 700 x g for 1 hour at 22 degrees celcius(Raoult et al., 1990). Following the spin, the inoculum was removed, the shell vial was washed with phosphate-buffered saline, medium was added, and the vial was incubated for 6 days at 37 degrees Celsius. The bacteria were visible in cells of the monolayer when Gimenez or immunofluorescent staining was performed(Maurin and Raoult, 1999). Subculturing: samples from shell vials were harvested and inoculated into 25-cm squared flasks containing HEL cell monolayers. The medium was changed every 3 days. When cells were determined to be heavily infected following examination by Gimenez staining, they were harvested and inoculated into 150-cm squared flasks(Raoult et al., 1990).
    2. Medium: Eagle's minimal essential medium (MEM) with 10% fetal calf serum and 1% glutamine(Raoult et al., 1990).
    3. Optimal Temperature: 37 degrees Celsius(Raoult et al., 1990).
  3. Culture of Phase I and Phase II Coxiella burnetii in murine macrophage-like cell lines :
    1. Description: Phase I Coxiella burnetii is the highly infectious, natural phase of the bacterium corresponding to smooth lipopolysaccharide (LPS). Phase I is found in infected animals, arthropods, or humans(Maurin and Raoult, 1999). Phase I Coxiella burnetii was found to proliferate in the murine macrophage-like tumor cell lines P388D1, J774, and PU-5-IR(Maurin and Raoult, 1999, Baca et al., 1981). Phase II is the "not very infectious" phase of Coxiella burnetii obtained only after serial passages in cultures. It corresponds to rough lipopolysaccharide (LPS)(Maurin and Raoult, 1999). Phase II C. burnetii proliferated in P388D1, J774, and PU-5-IR cell lines plus WEHI-3 and WEHI-274(Baca et al., 1981). Before exposure to the bacteria, cell lines were washed and suspended to appropriate concentrations in antibiotic-free medium. Following infection, the cells were kept at 37 degrees Celsius in a 4% CO2 atmosphere and passaged every 3 days. Cell-associated bacteria were visualized using Gimenez staining, (see protocol for Gimenez staining in "light microscopy, diagnostic test")(Baca et al., 1981).
    2. Medium: Dulbecco's modified Eagle's medium (DME) with 5-10% fetal calf serum with nonessential amino acids included for the PU-5 line only(Baca et al., 1981).
    3. Optimal Temperature: 37 degrees Celsius(Baca et al., 1981).
  4. Culture of Coxiella burnetii in BGM cells :
    1. Description: Coxiella burnetii was grown in Buffalo green monkey cells (BGM, Flow Laboratories). Confluent cell layers were infected with the bacteria and incubated at 37 degrees Celsius. Fresh medium was added after 20-24 hours. C. burnetii could be collected from the media of actively growing cultures after 7-8 days using differential centrifugation (8 minutes/4,000 x g/4 degrees Celsius to remove cell debris and 20 minutes/25,000 x g/4 degrees Celsius to collect the bacteria)(Macellaro et al., 1998).
    2. Medium: Eagle's minimal essential medium (MEM), supplemented with Earle's salts, 2 mM L-glutamine, 0.2% NaHCO3, 5% calf serum, and 1% nonessential amino acids(Macellaro et al., 1998).
    3. Optimal Temperature: 37 degrees Celsius(Macellaro et al., 1998).
  5. Culture of Coxiella burnetii in L929 mouse fibroblasts :
    1. Description: Coxiella burnetii was cultured in L929 mouse fibroblasts. After 1 week, L929 cells were sonicated and bacteria could be collected(Capo et al., 1999).
    2. Medium: Antibiotic-free minimal essential medium (MEM) supplemented with 4% fetal calf serum and 2 mM L-glutamine(Capo et al., 1999).
Epidemiology Information:
  1. Outbreak Locations:
    1. Q fever (caused by Coxiella burnetii) has been described in almost every country, with New Zealand remaining an exception(Maurin and Raoult, 1999). Explosive epidemics occur in stockyards, meat packing plants, and medical labs using sheep for research(Website 1).
  2. Transmission Information:
    1. From: Domestic animals , To: Domestic animals (Marrie and Raoult, 1997)
      Mechanism: Dairy cows are important in the spread of Q fever while beef cows are rarely infected. Once Coxiella burnetii is introduced into a herd, rapid spread occurs so that 80% of cows are positive within a few months. Infected wildlife may be important in infecting cattle since in a large portion of newly infected herds, a source of infection is not found(Marrie and Raoult, 1997).
    2. From: Ticks , To: Domestic animals , With Destination:Homo sapiens (Maurin and Raoult, 1999, Website 1)
      Mechanism: Ticks infect domestic and wild animals by expelling heavy loads of the organism with their feces onto the animal's skin at the time of feeding, however ticks are not considered essential in the natural cycle of Coxiella burnetti infection in livestock(Maurin and Raoult, 1999). Infected ticks have been found on rabbits, goats, cattle, sheep, and many other animals(Marrie and Raoult, 1997). Cattle, goats, and sheep are considered the primary reservoirs from which human contamination occurs most often by inhalation of infected fomites(Maurin and Raoult, 1999, Marrie and Raoult, 1997).
    3. From: Domestic animals , To: Homo sapiens (Maurin and Raoult, 1999, Norlander, 2000, Website 1)
      Mechanism: The aerosol route (inhalation of infected fomites) is the primary mode of human contamination with Coxiella burnetii(Maurin and Raoult, 1999). This can occur following contact with infected animals and their birth products, with wool from infected sheep, with infected straw or fertilizer, or with the laundry of a person exposed to the infected animal(Maurin and Raoult, 1999, Norlander, 2000, Website 1). The mode of spread results in the following persons being at risk for infection: abattoir workers, farmers, and veterinarians(Marrie, 1990). The largest outbreak of acute Q fever in the United Kingdom occurred in 1989. The most likely route of infection for the 147 diagnosed cases was assumed to be the windborne spread from farmland to an urban area(Norlander, 2000).
    4. From: Milk , To: Homo sapiens (Maurin and Raoult, 1999, Marrie and Raoult, 1997)
      Mechanism: There is a suggestion from epidemiological studies that ingestion of contaminated milk is a risk factor for Q fever infection. However, evidence from experiments where contaminated milk was fed to volunteers is contradictory, making this mode of infection controversial(Maurin and Raoult, 1999, Marrie and Raoult, 1997).
    5. From: Infected tick, intradermal inoculation, blood transfusion. , To: Homo sapiens (Marrie, 1990, Marrie and Raoult, 1997)
      Mechanism: There has been a report that a human became infected with Coxiella burnetii after crushing a tick between his fingers, developing Q fever 16 days later. However, aerosol exposure in this case could not be ruled out. In addition, a study in which volunteers were intradermally infected with C. burnetii all developed clinical signs of Q fever. There has also been one report of transmission of Q fever via a blood transfusion(Marrie, 1990, Marrie and Raoult, 1997).
    6. From: Homo sapiens , To: Homo sapiens (Milazzo et al., 2001)
      Mechanism: There have only been a few cases of documented person to person transmission of Q fever. There are two reports of transmission of Q fever to attendants during human autopsies, and one report of transmission of infection from a patient to hospital staff(Marrie and Raoult, 1997). Sexual Transmission: Although rare, sexual transmission of Q fever in humans has been documented. In a 1996 report, sheep shearers that acquired Q fever from sheep subsequently infected their wives via sexual intercourse. Another report in 2001 described sexually transmitted Q fever from a man (infected via the aerosol route) to his wife. This report demonstrated the presence of Coxiella burnetii in the man's semen, however, sexual intercourse may also involve the exchange of fluids such as saliva, blood, or urine, which may actually be the vehicle of infection(Milazzo et al., 2001). From Placenta to Fetus: Because Coxiella burnetii has been isolated from human placentas, it has been suggested that there may be a reactivation of infection at the time of pregnancy in women previously infected with the organism. This has rarely been documented however, and the overall importance of vertical transmission is thought to be unimportant in the overall epidemiology of Q fever in man(Marrie, 1990, Marrie and Raoult, 1997).
  3. Environmental Reservoir:
    1. Wild animals(Maurin and Raoult, 1999, Norlander, 2000):
      1. Description: Many species of wildlife including rats, mice, rabbits, water buffalo, bandicoots, hedgehogs, camels, baboons, ducks, geese, pigeons, and bats can be infected with Coxiella burnetti(Maurin and Raoult, 1999, Website 1, Norlander, 2000, Marrie and Raoult, 1997).
    2. Domestic animals(Maurin and Raoult, 1999, Norlander, 2000):
      1. Description: Cattle, sheep and goats are considered to be the main reservoirs of the agent responsible for infection of humans(Norlander, 2000). Cats and dogs may also represent reservoirs of Coxiella burnetii. The possibility of human Q fever acquired from infected dogs has been reported and human Q fever cases were described in Nova Scotia after contact with parturient cats(Maurin and Raoult, 1999). 44 (14.2%) of 310 pet cats in Japan were seropositive, as were 15 (41.7%) of 36 stray cats in Japan and 10 (8.6%) of 116 pet cats in Korea. The positivity among stray cats was significantly higher than among pet cats in both countries (P<0.01), but there was no significant difference in the positivity rate between Japan and Korea. Only 4 (1.3%) of pet cats in Japan were PCR-positive(Komiya et al., 2003).
    3. Ticks(Maurin and Raoult, 1999, Norlander, 2000):
      1. Description: Over 40 tick species are naturally infected with Coxiella burnetii and they expel heavy loads of C. burnetii with their feces onto the skin of the animal host at the time of feeding. 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 (Norlander, 2000). The possibility of C. burnetii being transmitted to humans via a tick bite has seldom been reported. Human Q fever, as opposed to other rickettsial diseases, is rarely, an arthropod-borne disease(Maurin and Raoult, 1999, Norlander, 2000).
    4. Dried sputum(Website 1):
      1. Description: dried sputum(Website 1).
      2. Survival: C. burnetii can survive for 30 days in dried sputum(Website 1).
    5. Soil(Website 1):
      1. Description: soil(Website 1).
      2. Survival: C. burnetii can survive for years within soil(Website 1).
    6. Dust(Website 1):
      1. Description: dust(Website 1).
      2. Survival: C. burnetii can survive for 120 days in dust(Website 1).
    7. Dried Urine(Website 1):
      1. Description: dried urine(Website 1).
      2. Survival: C. burnetii can survive for 49 days in the dried guinea pig urine(Website 1).
    8. Tick Feces(Website 1):
      1. Survival: C. burnetii can survive for 586 days in tick feces(Website 1).
    9. Milk(Website 1):
      1. Description: milk(Website 1).
      2. Survival: C. burnetii can survive for 42 months in milk stored at 4-6 degrees Celcius(Website 1).
    10. Wool(Website 1):
      1. Description: wool(Website 1).
      2. Survival: C. burnetii can survive for 12-16 months in wool kept at a temperature of 4-6 degrees Celcius(Website 1).
    11. Extracellular Environment(Heinzen et al., 1999):
      1. Description: extracellular environment(Heinzen et al., 1999).
      2. Survival: Coxiella burnetii can survive heating at 63 degrees Celcius for 30 minutes, exposure to a 10% salt solution for 180 days at room temperature, or exposure to 0.5% formalin for 24 hours(Heinzen et al., 1999).
  4. Intentional Releases:
    1. Intentional Release Information:
      1. Description: Q fever is deemed a category B biological terrorist agent, since, although it has potential for large-scale dissemination, it lacks capacity for the massive fatalities potentially caused by category A agents (smallpox, anthrax, plague, botulism, tularaemia, and viral haemorrhagic fevers). However, compared with category A agents, Q fever might be more suitable for use as a biological weapon because of its widespread availability, natural potential for aerosolised use, environmental stability, and the possibility of producing large quantities of infectious material(Website 4). Humans acquire Q fever by inhaling Coxiella burnetii(Website 4). Ease of aerosol dissemination, environmental persistence, and high infectivity (ID50 = 1-10) make C. burnetii a serious threat for military personnel and civilians. This agent has already been weaponized and mass-produced under various biological warfare programs(Seshadri et al., 2003). As an agent of biological warfare, Q fever is an incapacitating agent and would not be expected to cause significant fatalities(Website 4).
      2. Emergency Contact: In 1999, Q fever became a notifiable disease in the United States, thus health care providers are required to report to State or local public health officials (Q fever)(Website 3).
      3. Delivery Mechanism: If Coxiella burnetii was intentionally released, it would likely be distributed as a cloud of C. burnetii-infected particles that would be inhaled(Website 4). If used against troops, loss of manpower can range from 23% to 77%, and operational efficiency can be severely impaired. If used as a biological weapon in a civilian population, the degree of infectivity may rival that of anthrax, and although associated mortality will be low, Q fever can cause extensive acute and chronic morbidity(Madariaga et al., 2003).
      4. Containment: Coxiella burnetii is highly infectious via the aerosol route and very stable in the environment, but it is possible to disinfect contaminated articles by using a 0.5% hypochlorite solution, 1% Lysol, or 5% hydrogen peroxide(Website 2, Website 4, Marrie et al., 2000).
Diagnostic Tests Information
  1. Organism Detection Test:
    1. Gimenez staining :
      1. Description: The organism appears as a short rod which is not stained by Gram staining but which is visible after Giemsa or Gimenez staining(Fournier et al., 1998). After Gimenez staining, macrophages containing large vacuoles full of the organism can be identified at 400x magnification (see Fig. 6, Maurin and Raoult, 1999). Protocol for Gimenez staining (Gimenez, 1964): Reagents:1. Prepare a stock solution of carbol basic fuschin by mixing 100 ml of 10% (w/v) basic fuschin in 95% ethanol, 250 ml of 4% (v/v) aqueous phenol, and 650 ml of distilled water. Keep the stock solution at 37 degrees C for 48 hours before use (stock solution is stable for at least 10 months). 2. A 0.1M sodium phosphate buffer solution at pH 7.45 is made by mixing 3.5 ml of 0.2 M NaH2PO4, 15.5 ml of 0.2 M Na2HPO4, and 19 ml of distilled water.3. The working solution of carbol basic fuschin is prepared by mixing 4 ml of stock solution with 10 ml of pH 7.45 buffer. This is immediately filtered, and filtered again before every stain.4. Other solutions: 0.8% malachite green oxalate, 4% aqueous Fe(NO3)3/9H2O, and 5% aqueous fast green FCF.Procedure: 1. Prepare the sample for staining. If necessary, make a very thin smear. 2. After air drying (with or without fixing by passing through a flame) the preparation is covered with carbol basic fushcin (working solution) and let stand 1 to 2 minutes. 3. Wash thoroughly with water and cover the preparation with the malachite green solution for 6-9 seconds. 4. Repeat step 3 by again washing and covering the preparation with the malachite green solution for 6-9 seconds.5. Wash thoroughly with water and dry the preparation with absorbent paper(Maurin and Raoult, 1999, Gimenez, 1964).
    2. Indirect immunofluorescence staining :
      1. Description: Indirect immunofluorescence staining was used by Muhlemann et al. (1995) to demonstrate the presence of Coxiella burnetii in heart valves of patients treated for Q fever endocarditis. The method used, involved the isolation of C. burnetii from heart valve tissue and inoculation of the bacteria into human fetal lung fibroblasts (MCR-5). MCR-5 cell monolayers were examined by indirect immunofluorescence using high titer anti-Coxiella burnetii human serum followed by sheep anti-human Ig and Evan's blue counterstaining(Muhlemann et al., 1995).
    3. Immunohistologic/Immunoperoxidase Staining (Fournier et al., 1998, Brouqui et al., 1994, Raoult et al., 1994):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The detection of Coxiella burnetii in tissues is especially informative in patients who are undergoing treatment for chronic Q fever (Fournier et al., 1998). Brouqui et al. (1994) examined the valves of 17 patients with Q fever endocarditis using a monoclonal antibody (Raoult, 1994) and immunohistochemical methods. The organisms were clustered as a single intracytoplasmic mass within mononuclear cells and usually occupied the entire cytoplasm(Fournier et al., 1998).
  2. Immunoassay Test:
    1. A monoclonal antibody-based capture ELISA/ELIFA :
      1. Time to Perform: 1-to-2-days
      2. Description: A monoclonal antibody-based capture ELISA/ELIFA for detection of Coxiella burnetii in clinical specimens was developed in 1992 by Thiele and coworkers. The assay used a monoclonal antibody (4/11) as capture and detection antibody. The system recognized a minimum dose of 2500 Coxiella burnetii particles, demonstrated broad reactivity with both phase I and phase II Coxiella burnetii, and did not cross-react with 20 other strains of bacteria(Thiele et al., 1992).
    2. PANBIO Coxiella burnetii (Q fever) IgM ELISA test :
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Serum antibodies of the IgM class, when present, combine with C. burnetii antigen attached to the polystyrene surface of the microwell test strips. Residual serum is removed by washing and peroxidase conjugated anti-human IgM added. The microwells are washed and a colourless substrate system, tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) is added. The substrate is hydrolysed by the enzyme and the chromogen changes to a blue color. After stopping the reaction with acid, the TMB becomes yellow. The color intensity is directly related to the concentration of the C. burnetii IgM antibodies in the test sample (Coxiella burnetti IgM). During acute Q fever, seroconversion is usually detected from 7 to 15 days after the onset of clinical symptoms and antibodies are detected by the third week in about 90% of cases(Maurin and Raoult, 1999, Website 14). A comparison of the IgM ELISA to indirect fluorescent antibody test (IFAT) and the complement fixation test (CFT) was performed by Field et al., 2000. The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (n = 6), chlamydia (n = 5), or legionella (n = 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PANBIO C. burnetii (Q fever) IgM ELISA (F = 187) is superior to that of CFT (F = 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever(Field et al., 2000, Website 14).
    3. PANBIO Coxiella burnetii (Q fever) IgG ELISA test :
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Serum antibodies of the IgG class, when present, combine with C. burnetii antigen attached to the polystyrene surface of the microwell test strips. Residual serum is removed by washing and peroxidase conjugated anti-human IgG added. The microwells are washed and a colourless substrate system, tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) is added. The substrate is hydrolysed by the enzyme and the chromogen changes to a blue color. After stopping the reaction with acid, the TMB becomes yellow. The color intensity is directly related to the concentration of the C. burnetii IgG antibodies in the test sample. In Q fever infection, IgG antibodies reach peak levels within six weeks and slowly decline to lower levels which persist indefinitely. A significant rise in specific IgG antibody suggests a recent infection(Field et al., 2002). In a study (Field et al., 2002), the PANBIO IgG ELISA was compared to the complement fixation test (CFT), and the indirect fluorescent-antibody test (IFAT) was used to resolve discrepant results between the other two tests. A total of 214 serum samples were tested. The ELISA demonstrated a specificity of 96% (46 of 48 samples) and a sensitivity of 71% (95 of 134 samples). Of the six serum pairs showing CFT seroconversion, three pairs showed a corresponding ELISA seroconversion. No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma, brucella, and chlamydia infections. One of the 13 patients with leptospirosis demonstrated a positive result in the ELISA but not in the CFT or the IFAT, and Legionella pneumophila serogroup 4 antibody was found in one of the two sera that were false-positive by ELISA. The results presented in this study suggest that the PANBIO Q fever IgG ELISA is a specific alternative method for prevaccination testing and an aid for the diagnosis of Q fever. This test is suitable for use as a screening assay, with CFT and/or IFAT used to confirm negative results(Field et al., 2002).
    4. IFA, microimmunofluorescence test :
      1. Description: The IFA remains the reference technique for Q fever diagnosis. The microimmunofluorescence test has the advantage of requiring only small amounts of antigen. In order to prepare antigens for this test, phase II C. burnetii Nine Mile reference strain is grown in confluent layers of L929 mouse fibroblasts, while phase I antigens are obtained from the spleens of mice inoculated with phase II organisms. This method of preparation has been demonstrated to yield antigens with the highest sensitivity for C. burnetii antibody detection. Sera are diluted in phosphate-buffered saline with 3% nonfat powdered milk to avoid the nonspecific fixation of antibodies. This method can be used to determine antibodies to phases I and II in the IgG, IgM, and IgA fractions. However, test results can be confounded by the presence of a rheumatoid factor. Thus, a rheumatoid factor absorbant is used in order to remove IgG before the determination of IgM and IgA. The choice of a negative cutoff titer depends upon the source and purity of the antigen and the amount of background antigen stimulation in the population to be studied. Screening is performed with anti-phase II anti-immunoglobulins with a 1:50 dilution for the tested sera. Positive sera are then serially diluted and tested for the presence of anti-phase I and II IgG, IgM, and IgA. Seroconversion to IgM is usually detected 7 to 15 days after the onset of clinical symptoms. About 90% of patients have detectable antibodies by the third week(Fournier et al., 1998).
    5. PANBIO Coxiella burnetii (Q fever) IFA slides :
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The PANBIO Coxiella burnetii IFA slides contain both Phase I and Phase II purified organisms as well as a normal yolk sac (NYS) control. All three are represented on each well of the slides as distinct microdots. Dilutions of the patient's serum are placed in wells on the slide, permitting the antibody to bind specifically to the organisms. Bound antibodies are tagged with a fluorescein labeled anti-human conjugate and observed using a fluorescence microscope. In this format, organisms are readily identified as small coccobacilli. Fluorescent coxiellae are bright yellow against a dull red background (counterstain) (Coxiella burnetti IFA)(Website 16). During acute Q fever, seroconversion is usually detected from 7 to 15 days after the onset of clinical symptoms and antibodies are detected by the third week in about 90% of cases(Maurin and Raoult, 1999).
    6. PANBIO DIP-S-TICKS :
      1. Time to Perform: minutes-to-1-hour
      2. Description: The PANBIO Q Fever Dip-S-Ticks assay utilizes an enzyme-linked immunoassay (EIA) dot technique for thedetection of antibodies to Coxiella burnetii. The antigens are dispensed as discrete dots onto a solid membrane. After adding specimen to a reaction vessel, an assay strip is inserted, allowing patient antibodies reactive with the test antigen to bind to the strip's solid support membrane. In the second stage, the reaction is enhanced by removal of non-specifically bound materials. During the third stage, alkaline phosphatase-conjugated anti-human antibodies are allowed to react with bound patient antibodies. Finally, the strip is transferred to enzyme substrate reagent, which reacts with bound alkaline phosphate to produce an easily seen, distinct spot. Cultivation of the Q fever organism, C. burnetii, produces two distinct antigens: Phase I and Phase II. Antibodies to Phase II antigen are produced early in acute infections. Later, Phase I antibodies can be detected but may be in reduced concentration with respect to Phase II. Patients having chronic infections exhibit high antibody levels to both Phase I and Phase II antigens. PANBIO Q fever Dip-S-Ticks have dots for both Phase I and Phase II antigens. Three dots are for Phase II (acute infection) determination and one is for Phase I (chronic infection) determination(Website 17).
  3. Nucleic Acid Detection Test: