Section VIII-D: Rickettsial Agents

Coxiella burnetii

Coxiella burnetii is the etiologic agent of Q fever. C. burnetii is a bacterial obligate

intracellular pathogen that undergoes its developmental cycle within an acidic
vacuolar compartment exhibiting many characteristics of a phagolysosome. The
developmental cycle consists of a large (approximately 1 µm in length) cell variant
that is believed to be the more metabolically active, replicative cell type and a
smaller, more structurally stable cell variant that is highly infectious and quite
resistant to drying and environmental conditions.1-4 The organism undergoes a
virulent (Phase I) to avirulent (Phase II) transition upon serial laboratory passage
in eggs or tissue culture.

The infectious dose of virulent Phase I organisms in laboratory animals

has been calculated to be as small as a single organism.5 The estimated human
infectious dose for Q fever by inhalation is approximately 10 organisms.6 Typically,
the disease manifests with flu-like symptoms including fever, headache, and
myalgia but can also cause pneumonia and hepatomegaly. Infections range from
sub-clinical to severe although primary infections respond readily to antibiotic
treatment. Although rare, C. burnetii is known to cause chronic infections such
as endocarditis or granulomatous hepatitis.7

Occupational Infections

Q fever is the second most commonly reported LAI in Pike’s compilation. Outbreaks
involving 15 or more persons were recorded in several institutions.8,9 Infectious
aerosols are the most likely route of laboratory-acquired infections. Experimentally
infected animals also may serve as potential sources of infection or laboratory
and animal care personnel. Exposure to naturally infected, often fasymptomatic
sheep and their birth products is a documented hazard to personnel.10,11

Natural Modes of Infection

Q fever (Q for query) occurs worldwide. Broad ranges of domestic and wild
mammals are natural hosts for Q fever and sources of human infection. Parturient
animals and their birth products are common sources of infection. The placenta
of infected sheep may contain as many as 109 organisms per gram of tissue12
and milk may contain 105 organisms per gram. The resistance of the organism to
drying and its low infectious dose can lead to dispersal from contaminated sites.

Laboratory Safety and Containment Recommendations

The necessity of using embryonated eggs or cell culture techniques for the
propagation of C. burnetii leads to extensive purification procedures. Exposure to
infectious aerosols and parenteral inoculation cause most infections in laboratory
and animal care personnel.8,9 The agent may be present in infected arthropods

Agent Summary Statements: Rickettsial Agents 195

and in the blood, urine, feces, milk, and tissues of infected animals or human
hosts. Exposure to naturally infected, often asymptomatic, sheep and their birth
products is a documented hazard to personnel.10,11 Recommended precautions
for facilities using sheep as experimental animals are described elsewhere.10,13

BSL-2 practices and facilities are recommended for nonpropagative laboratory

procedures, including serological examinations and staining of impression smears.
BSL-3 practices and facilities are recommended for activities involving the inoculation,
incubation, and harvesting of embryonated eggs or cell cultures, the necropsy of
infected animals and the manipulation of infected tissues. Experimentally infected
animals should be maintained under ABSL-3 because infected rodents may shed the
organisms in urine or feces.8 A specific plaque-purified clonal isolate of an avirulent
(Phase II) strain (Nine Mile) may be safely handled under BSL-2 conditions.14

Special Issues

Vaccines  An investigational Phase I, Q fever vaccine (IND) is available on a

limited basis from the Special Immunizations Program (301-619-4653) of the
USAMRIID, Fort Detrick, Maryland, for at-risk personnel under a cooperative
agreement with the individual’s requesting institution. The use of this vaccine
should be restricted to those who are at high risk of exposure and who have no
demonstrated sensitivity to Q fever antigen. The vaccine can be reactogenic in
those with prior immunity, thus requires skin testing before administration. The
vaccine is only administered at USAMRIID and requires enrollment in their Q
fever IND Immunization Program. For at-risk laboratory workers to participate in
this program, fees are applicable. Individuals with valvular heart disease should
not work with C. burnetii. (See Section VII.)

Select Agent  C. burnetii is a select agent requiring registration with CDC

and/or USDA for possession, use, storage and/or transfer. See Appendix F
for additional information.

Transfer of Agent  Importation of this agent may require CDC and/or USDA
importation permits. Domestic transport of this agent may require a permit from
USDA/APHIS/VS. A DoC permit may be required for the export of this agent to
another country. See Appendix C for additional information.

Rickettsia prowazekii; Rickettsia typhi (R. mooseri); Orientia (Rickettsia)

tsutsugamushi and Spotted Fever Group agents of human disease;
Rickettsia rickettsii, Rickettsia conorii, Rickettsia akari, Rickettsia australis,
Rickettsia siberica, and Rickettsia japonicum

Rickettsia prowazekii, Rickettsia typhi (R. mooseri), Orientia (Rickettsia) tsutsugamushi

and the Spotted Fever Group agents of human disease (Rickettsia rickettsii,
Rickettsia conorii, Rickettsia akari, Rickettsia australis, Rickettsia siberica, and

196 Biosafety in Microbiological and Biomedical Laboratories

Rickettsia japonicum) are the etiologic agents of epidemic typhus, endemic (murine)
typhus), scrub typhus, Rocky Mountain spotted fever, Mediterranean spotted fever,
rickettsialpox, Queensland tick typhus, and North Asian spotted fever, respectively.

Rickettsia spp. are bacterial obligate intracellular pathogens that are

transmitted by arthropod vectors and replicate within the cytoplasm of eukaryotic
host cells. Two groups are recognized within the genus, the typhus group and
the spotted fever group. The more distantly related scrub typhus group is now
considered a distinct genus, Orientia. Rickettsiae are primarily associated with
arthropod vectors in which they may exist as endosymbionts that infect mammals,
including humans, through the bite of infected ticks, lice, or fleas.15

Occupational Infections

Pike reported 57 cases of laboratory-associated typhus (type not specified), 56

cases of epidemic typhus with three deaths, and 68 cases of murine typhus.8
Three cases of murine typhus have been reported from a research facility.16 Two
were associated with handling of infectious materials on the open bench; the third
case resulted from an accidental parenteral inoculation. These three cases
represented an attack rate of 20% in personnel working with infectious materials.
Rocky Mountain spotted fever is a documented hazard to laboratory personnel.
Pike reported 63 laboratory-associated cases, 11 of which were fatal.8 Oster
reported nine cases occurring over a six-year period in one laboratory. All were
believed to have been acquired because of exposure to infectious aerosols.17

Natural Modes of Infection

The epidemiology of rickettsial infections reflects the prevalence of rickettsiae

in the vector population and the interactions of arthropod vectors with humans.
Epidemic typhus is unusual among rickettsiae in that humans are considered the
primary host. Transmission is by the human body louse; thus, outbreaks are now
associated with breakdowns of social conditions. Endemic typhus is maintained
in rodents and transmitted to humans by fleas. The various spotted fever group
rickettsiae are limited geographically, probably by the distribution of the arthropod
vector, although specific spotted fever group rickettsiae are found on all continents.15

Laboratory Safety and Containment Recommendations

The necessity of using embryonated eggs or cell culture techniques for the
propagation of Rickettsia spp. incorporates extensive purification procedures.
Accidental parenteral inoculation and exposure to infectious aerosols are the
most likely sources of LAI.18 Aerosol transmission of R. rickettsii has been
experimentally documented in nonhuman primates.19 Five cases of rickettsialpox
recorded by Pike were associated with exposure to bites of infected mites.8
Naturally and experimentally infected mammals, their ectoparasites, and their
infected tissues are potential sources of human infection. The organisms are
relatively unstable under ambient environmental conditions.

Agent Summary Statements: Rickettsial Agents 197

 BSL-2 practices, containment equipment, and facilities are recommended
for nonpropagative laboratory procedures, including serological and fluorescent
antibody procedures, and for the staining of impression smears. BSL-3 practices,
containment equipment, and facilities are recommended for all other manipulations
of known or potentially infectious materials, including necropsy of experimentally
infected animals and trituration of their tissues, and inoculation, incubation, and
harvesting of embryonated eggs or cell cultures. ABSL-2 practices, containment
equipment, and facilities are recommended for the holding of experimentally
infected mammals other than arthropods. BSL-3 practices, containment equipment,
and facilities are recommended for animal studies with arthropods naturally or
experimentally infected with rickettsial agents of human disease. (See Appendix E.)

Several species, including R. montana, R. rhipicephali, R. belli, and R. canada,

are not known to cause human disease and may be handled under BSL-2 conditions.
New species are being described frequently and should be evaluated for appropriate
containment on a case-by-case basis. Because of the proven value of antibiotic
therapy in the early stages of ricketsial infection, it is essential that laboratories
have an effective system for reporting febrile illnesses in laboratory personnel,
medical evaluation of potential cases and, when indicated, institution of
appropriate antibiotic therapy.

Special Issues
Medical Response  Under natural circumstances, the severity of disease caused
by rickettsial agents varies considerably. In the laboratory, very large inocula are
possible, which might produce unusual and perhaps very serious responses.
Surveillance of personnel for laboratory-associated infections with rickettsial
agents can dramatically reduce the risk of serious consequences of disease.
Experience indicates that infections adequately treated with specific anti-rickettsial
chemotherapy on the first day of disease do not generally present serious
problems. However, delay in instituting appropriate chemotherapy may result
in debilitating or severe acute disease ranging from increased periods of
convalescence in typhus and scrub typhus to death in R. rickettsii infections.
The key to reducing the severity of disease from laboratory-associated infections
is a reliable medical response which includes: 1) round-the-clock availability of
an experienced medical officer; 2) indoctrination of all personnel on the potential
hazards of working with rickettsial agents and advantages of early therapy; 3) a
reporting system for all recognized overt exposures and accidents; 4) the reporting
of all febrile illnesses, especially those associated with headache, malaise, and
prostration when no other certain cause exists; and 5) an open and non-punitive
atmosphere that encourages reporting of any febrile illness.

Select Agent  R. prowazekii and R. rickettsii are select agents requiring

registration with CDC and/or USDA for possession, use, storage and/or transfer.
See Appendix F for additional information.

198 Biosafety in Microbiological and Biomedical Laboratories

Transfer of Agent  Importation of this agent may require CDC and/or USDA
importation permits. Domestic transport of this agent may require a permit from
USDA/APHIS/VS.

References

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2. Ignatovich VF. The course of inactivation of Rickettsia burnetii in fluid media.
Zh Mikrobiol Epidemiol Immunobiol. 1959;30:134-41.
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Dis. 1987;9:935-46.
4. Heinzen RA, Hackstadt T, Samuel JE. Developmental biology of Coxiella
burnetii. Trends Microbiol. 1999;7:149-54.
5. Ormsbee R, Peacock M, Gerloff R, et al. Limits of rickettsial infectivity. Infect
Immun. 1978;19:239-45.
6. Wedum AG, Barkley WE, Hellman A. Handling of infectious agents. J Am Vet
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8. Pike RM. Laboratory-associated infections: Summary and analysis of 3,921
cases. Health Lab Sci. 1976;13:105-14.
9. Johnson JE, Kadull PJ. Laboratory-acquired Q fever. A report of fifty cases.
Am J Med. 1966;41:391-403.
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controlling a sheep zoonosis in a lab animal facility. Lab Anim. 1981;10:24-7.
11. Meiklejohn G, Reimer LG, Graves PS, et al. Cryptic epidemic of Q fever in
a medical school. J Infect Dis. 1981;144:107-13.
12. Welsh HH, Lennette EH, Abinanti FR, et al. Q fever in California IV.
Occurrence of Coxiella burnetii in the placenta of naturally infected sheep.
Public Health Rep. 1951;66:1473-7.
13. Bernard KW, Helmick CG, Kaplan JE, et al. Q fever control measures:
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14. Hackstadt T. Biosafety concerns and Coxiella burnetii. Trends Microbiol.
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15. Hackstadt T. The biology of rickettsiae. Infect Agents Dis. 1996;5:127-43.
16. Centers for Disease Control and Prevention. Laboratory-acquired endemic
typhus. MMWR Morb Mortal Wkly Rep. 1978;27:215-6.
17. Oster CN, Burke DS, Kenyon RH, et al. Laboratory-acquired Rocky
Mountain spotted fever. The hazard of aerosol transmission. N Engl J Med.
1977;297:859-63.
18. Hattwick MA, O’Brien RJ, Hanson BF. Rocky Mountain spotted fever:
epidemiology of an increasing problem. Ann Intern Med. 1976;84:732-9.
19. Saslaw S, Carlisle HN. Aerosol infection of monkeys with Rickettsia rickettsii.
Bacteriol Rev. 1966;30:636-45.

Agent Summary Statements: Rickettsial Agents-References 199