Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Rickettsia rickettsii (Website 1):
      1. GenBank Taxonomy No.: 783
      2. Description: R. ricketsii is native to the New World and causes the malady known as Rocky Mountain spotted fever (RMSF). RMSF is transmitted by the bit of an infected tick while feeding on warm-blooded animals, including humans. Man is an accidental host in the rickettsia-tick life cycle and is not required to maintain the rickettsiae in nature(Weiss, 1988). Wolbach deserves the credit for the first detailed description of the etiologic agent in 1919. He clearly recognized it as an intracellular bacterium which was seen most frequently in endothelial cells. He was struck by the fact that in the tick, and also in mammalian cells, the microorganism was intranuclear. The nucleus was often completely filled with minute particles and often was distended. Although Wolbach recognized its similarity to the agent of typhus and tsutsugamushi fever, he did not regard the designation 'rickettsia' as appropriate. He proposed the name Dermacentroxenus rickettsi. Brumpt felt that the etiologic agent of RMSF, despite some uncertainty about its properties, belonged in the genus Rickettsia and in 1922 proposed the name Rickettsia rickettsi(Weiss, 1988B).

1. R. rickettsii life cycle
   1. Stage Information:
      1. Rickettsia rickettsii vegetative state:
         • Size: R. rickettsii, the causative agent of RMSF, is a small (0.2 to 0.5 by 0.3 to 2.0 um), obligate, intracellular bacterium.
         • Shape: Ricketts described small diplococcoid bodies or diplobacilli in infected animals, particularly in tick eggs.
         • Picture(s):
           • Rickettsia rickettsii (Website 3)
             
             
             
             Description: Gimenez stain of tick hemolymph cells infected with R. rickettsii(Website 3).
   2. Picture(s):
      • Rickettsial rickettsii (Website 2)
        
        
        
        Description: Life cycle of Rickettsia rickettsii in its tick and mammalian hosts(Website 2).
   3. Description: Rickettsia rickettsii are maintained in nature by transstadial passage within, and transovarial (vertical) transmission between generations of ixodid ticks. These ticks also vector R. rickettsii to and from various rodent reservoirs and other small mammals. Naive larval and nymphal ticks become infected while feeding on small rodents (eg, mice, voles, squirrels, or chipmunks) with acute rickettsemia. To enable transovarial transmission, female ticks need to ingest numerous rickettsiae or be infected transtadially. Male ticks can transfer R. rickettsii to females during the mating process via spermatozoa or other body fluids, thus contributing to the maintenance of the organism from one generation to another. Ticks can remain infective for life (possibly 2 to 5 years), especially if there are long periods between blood feeding. Ticks transmit R. rickettsii to a vertebrate principally during their feeding behavior. However, human infection has occurred much less often following transdermal or inhalation exposure to tick fluids (hemolymph), tick feces, or crushed tick tissues. In the natural history of R. rickettsii transmission, human and domestic dog infections are considered incidental events(Warner and Wallace, 2002).

1. Genome of Rickettsia rickettsii
   1. Description: .
   2. Chromosome
      1. GenBank Accession Number: Website5
      2. Size: 1257710 bp(Website 5).

1. General biosafety information
   1. Level: 2 and 3.
   2. Precautions: Biosafety Level 2 practices and facilities are recommended for nonpropagative laboratory procedures, including serological and fluorescent antibody procedures, and for the staining of impression smears. Biosafety Level 3 practices 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 embryonate eggs or cell cultures. Animal Biosafety Level 2 practices and facilities are recommended for the holding of experimentally infected mammals other than arthropods. Level 3 practices and facilities are recommended for animal studies with arthropods naturally or experimentally infected with rickettsial agents of human disease(Website 12).

1. R. rickettsii culture techniques :
   1. Description: Isolation of rickettsiae in cell culture can be achieved in 4 to 7 days in many cases, but use of antibiotic-free cell culture is mandatory because most antimicrobial agents inhibit rickettsial growth in virto. The reliance on antimicrobial agents in the medium to cover up for minor lapses in technique and contamination with normal flora is virtually universal. Cultivation of rickettsiae depends on meeting the technical challenge of antimicrobial agent-free cell culture. The work should be performed in a biohazard containment safety cabinet in a room under relative negative pressure with an anteroom. The worker must wear mask, gloves, and gown. R. rickettsii can be isolated from blood, plasma and tissues in Vero cells, L cells, primary chicken fibroblasts, and other primary and continuous cell lines. The shell vial centrifugation method offers and attractive approach. Rickettsiae can also be isolated by inoculation of the yolk sac of antiobiotic-free, 5- to 6-day-old embryonated hen eggs. Cultivated rickettsiae are identified provisionally by stianing with immunofluorescence and the Gimenez method(Walker, 1989).

1. Outbreak Locations:
   1. Currently no epidemic outbreak information is available.
2. Transmission Information:
   1. From: Ticks , To: Ticks (Warner and Wallace, 2002)
      Mechanism: Rickettsia rickettsii are maintained in nature by transstadial passage within, and transovarial (vertical) transmission between generations of ixodid ticks(Warner and Wallace, 2002). To enable transovarial transmission, female ticks need to ingest numerous rickettsiae or be infected transtadially. Male ticks can transfer R. rickettsii to females during the mating process via spermatozoa or other body fluids, thus contributing to the maintenance of the organism from one generation to another(Warner and Wallace, 2002).
   2. From: Ticks , To: Mammals
      Mechanism: Eggs from a female tick infected with R. rickettsii develop into infected larvae, which then feed on and infect small rodents with rickettsiae that are present in their saliva(McDade and Newhouse, 1986). Larvae infected with R. rickettsii molt to become infected nymphs that can infect medium-sized animals while feeding. After the nymphs molt, the resulting adult ticks can also infect larger animals(McDade and Newhouse, 1986). Rickettsiae are inoculated into the pool of blood in the dermis from which the tick is feeding. After the tick has fed for 6 hours, some rickettsiae, along with digestive secretions, are released from its salivary glands. Some ticks feed for even longer times before inoculation rickettsiae into the host's skin(Walker, 1995).
   3. From: Mammals , To: Ticks
      Mechanism: Naive larval and nymphal ticks become infected while feeding on small rodents (eg, mice, voles, squirrels, or chipmunks) with acute rickettsemia(Warner and Wallace, 2002).
   4. From: Human , To: Human
      Mechanism: Cases have been rarely acquired by laboratory personnel through direct inoculation or inhalation of aerosols containing R. rickettsii, by transfusion, or by needlestick injury from an infected patient(Weber and Walker, 1991). Aerosol infection from dried feces is unlikely because R. rickettsii loses its infectiousness rather rapidly in such material. Nevertheless, infections via the respiratory tract have been reported, especially among laboratory personnel(Burgdorfer, 1988).
3. Environmental Reservoir:
   1. Ticks:
      1. Description: Rocky Mountain spotted fever is a vector-borne disease transmitted by certain species of ticks that also serve as the reservoir or natural hosts(Weber and Walker, 1991). Even in highly endemic ares, such as the southeastern coastal states of Virginia and North Carolina, and Oklahoma, only a few ticks are infected with R. rickettsii(Sexton and Kaye, 2002).
      2. Survival: In its tick vectors, R. rickettsii produces a generalized infection that persists throughout the life span of the tick. Hemocytes and tissues of the hypodermis, Malpighian tubules, salivary glands, and digestive and genital systems usually have moderate to heavy infections that often become massive as a result of intense rickettsial multiplication during the feeding process of the tick(Burgdorfer, 1988). One should not conclude, however, that R. rickettsii and its acarine hosts have developed a perfect symbiotic relationship, or that infections of various tick species with R. rickettsii are always generalized, continuous, or mutually beneficial. Some tick infections are incomplete and cannot sustain the transmission of R. rickettsii. Other infections, although complete, can result in decreased fecundity or viability, or even death of the tick. For example, in established hosts like Dermacentor variabilis and D. andersoni high mortality rates were observed among naturally infected engorged female ticks beginning with the fifth filial generation, and these infected females also began to deposit less than half the number of eggs deposited by uninfected ticks. It should be noted, however, that 100% filial infection was observed among the viable ticks through 12 generations of infected D. andersoni, despite the high tick mortality(McDade and Newhouse, 1986). Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88. 3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4 C had a lower mortality (80.9%) than did those held at 21 C (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect of R. rickettsii may explain the low prevalence of infected ticks in nature and affect its enzootic maintenance(Niebylski et al., 1999).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: .
      2. Containment: Biosafety Level 2 practices and facilities are recommended for nonpropagative laboratory procedures, including serological and fluorescent antibody procedures, and for the staining of impression smears. Biosafety Level 3 practices 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 embryonate eggs or cell cultures. Animal Biosafety Level 2 practices and facilities are recommended for the holding of experimentally infected mammals other than arthropods. Level 3 practices and facilities are recommended for animal studies with arthropods naturally or experimentally infected with rickettsial agents of human disease(Website 32).

1. Organism Detection Test:
   1. Indirect immunofluorescence assay (IFA) :
      1. Time to Perform: unknown
      2. Description: Serologic assays are the most widely available and frequently used methods for confirming cases of Rocky Mountain spotted fever. The indirect immunofluorescence assay (IFA) is generally considered the reference standard in Rocky Mountain spotted fever serology and is the test currently used by CDC and most state public health laboratories. IFA can be used to detect either IgG or IgM antibodies. Blood samples taken early (acute) and late (convalescent) in the disease are the preferred specimens for evaluation. Most patients demonstrate increased IgM titers by the end of the first week of illness. Diagnostic levels of IgG antibody generally do not appear until 7-10 days after the onset of illness. It is important to consider the amount of time it takes for antibodies to appear when ordering laboratory tests, especially because most patients visit their physician relatively early in the course of the illness, before diagnostic antibody levels may be present. The value of testing two sequential serum or plasma samples together to show a rising antibody level is considerably more important in confirming acute infection with rickettsial agents because antibody titers may persist in some patients for years after the original exposure(Website 4). The IFA is performed on whole rickettsiae, which contain a vast array of protein and carbohydrate antigens. The organisms are affixed to microscopic slides and are reacted with serial dilutions of serum. The presence of antibody attached to the rickettsia is detected by fluorescein-conjugated antibodies to human immunoglobulins. Variations in the technique have included detection of antibody of immunoglobulin M class and demonstration of antibodies by immunoperoxidase instead of immunofluorescence. The availability of an ultraviolet microscope is required for the IFA test. Variations in the endpoint titer may occur because the anti-immunoglobulin conjugate, and the skill of the microscopist. The IFA is generally accepted as the best serologic test presently available, the one with which new assays should be compared(Walker, 1989). The IFA is generally considered to be the gold standard, although cross-reaction with other spotted fever group rickettsiae may occur even with this method. Despite its superior sensitivity and specificity, the IFA is inconvenient. Generally, it can be performed only at public health laboratories or other research or commercial reference facilities(Silber, 1996).
      3. Picture(s):
         • IFA reaction (Website 4)
           
           
           
           Description: IFA reaction of a positive human serum on Rickettsia rickettsii grown in chicken yolk sacs, 400X. Copyright: CDC(Website 4).
   2. Plaque Assay :
      1. Time to Perform: unknown
      2. Description: Primary cultures of human umbilical vein endothelial cells were inoculated with plaque-purified Rickettsia rickettsii. After adsorption of rickettsiae, monolayers were overlaid with medium containing 0.5% agarose. Small plaques appeared on day 4 postinoculation, and distinct 1- to 2-mm plaques were observed on day 5. Plaquing efficiency was less than that of primary chicken embryo cells in the same medium. Human endothelial cell monolayers were susceptible to infection by R. rickettsii and underwent necrosis as demonstrated by supravital staining. The topographic association of endothelial cell necrosis and rickettsial infection in the plaque model confirmed the direct cytopathic effect of R. rickettsii on human endothelium. Uninfected cells appeared normal by supravital staining and transmission electron microscopy. This model offers the possibility of investigating rickettsial pathogenesis and mechanisms of enhanced severity of Rocky Mountain spotted fever in specific genetically determined conditions(Walker et al., 1982). A plaque technique for the assay of Rickettsia rickettsii is described. The method employs primary chick or green monkey kidney monolayer cell cultures with either an agarose or special Noble agar overlay. Plaques were counted in 6 days and resultant titers correlated well with LD50 end points obtained by a standard assay in embryonated eggs. Identification of the plaque-forming organisms was accomplished by direct observation of rickettsiae-like bodies in the monolayer lesions, inhibition of plaques by antibiotics, sensitivity of plaques to specific immune serum, and failure to cultivate other microorganisms from the infected cells. Versatility of the test was demonstrated by assaying samples of rickettsiae from several different sources commonly used in our laboratory. These included infected yolk sacs, various cell cultures, and infected guinea pig tissue. Sufficient numbers of viable rickettsiae were present in the cells of a single lesion to permit direct recovery(Weinberg et al., 1969).
   3. Immunoperoxidase Staining of Parafin-Embedded Tissue :
      1. Time to Perform: 1-to-2-days
      2. Description: Immunoperoxidase (IP) staining is also an effective means of staining R. rickettsii. Immunoperoxidase methods can be applied with relative ease to paraffin-embedded biopsies. Tissues stained by the IP method can be examinied by conventional bright-field microscopy. Although paraffin embedding and peroxidase staining are more time-consuming than IF staining of frozen sections, new rapid embedding and IP staining protocols may achieve conclusive results in less than 24 hours(Procop et al., 1997). The IP staining method, although not as rapid as the IF method, is usually available on a next-day basis in most pathology laboratories(Procop et al., 1997).
      3. False Negative: Three patients had false negative IP results(Procop et al., 1997).
   4. Immunofluorescence :
      1. Time to Perform: unknown
      2. Description: Not until 1976 were spotted fever rickettsiae identified by immunofluorescence diagnostically in cutaneous biopsies and autopsy tissues. Extensive experience with this acute diagnostic approach led to its use as a routing procedure in some medical centers. Best results are obtained by selection of a classic petechial lesion centered in an erythematous maculopapule and removal of this sample of skin with a 3 mm punch under local anesthesia. The sample is mounted in polyethylene glycol. Frozen sections are cut perpendicular to the epidermal layer at 4 um thickness at 5 to 12 levels extending through the petechia or through two-thirds of the tissue of a nonpetechial lesion. After fixation in absolute acetone for 10 min and air drying, the sections are reacted with an anti-spotted fever group rickettsial antibody conjugated with fluorescein isothiocyanante for 30 min, and mounted under a cover slip for fluorescence microscopy. A well-characterized conjugate prepared at the CDC has been in standard use for 13 years(Walker, 1989). RMSF is diagnosed when three or more fluorescent structures compatible with rickettsiae are identified in the blood vessel wall in the dermis. With experience, the test is highly specific (100%) and sufficiently sensitive (70%) to be useful in making clinical decisions(Walker, 1989).
      3. False Negative: False-negative results increase after 48 h of antirickettsial treatment(Walker, 1989).
   5. Intraperitoneal inoculation of Guinea pigs :
      1. Time to Perform: unknown
      2. Description: Institutions with facilities for handling animals infected with biohazard class 3 agents can isolate R. rickettsii from human blood or tissues by intraperitoneal inoculation of adult male guinea pigs. Infected animals develop fever of more than 40 C and rickettsia-induced edemea of the scrotum, which sometimes proceeds to hemorrhage and necrosis. Rickettsiae are demonstrable in smears and frozen sections of tunica vaginalis, epididymis, and spleen within days after the onset of fever in the animal. Animals that survive are bled, and convalescent sera are examined for antibodies to R. rickettsii. For major medical centers in endemic regions, maintenance of a few adult male guinea pigs during May to September for isolation of R. rickettsii offers the opportunity to make the definitive diagnosis of RMSF. Reluctance to work with animals impairs the ability to investigate infectious diseases(Walker, 1989). It is important to maintain sensitive methods of recovering fastidious microorganisms such as rickettsiae, even if they are quite old-fashioned. It is erroneous to believe that they have been supplanted by other more sensitvie methods(Walker, 1989).
   6. Geimsa or Gram stain for light microscopy :
      1. Time to Perform: unknown
      2. Description: After the development of the Giems method for demonstraction of R. rickettsii by Wolbach, for three decades there was no pressure to apply the technique as an actue diagnostic test because there was no effective treatment. Subsequently, the histologic demonstration of rickettsia by Giemsa stain in infected tissues has become virtually a lost art. The Brown-Hopps method, a tissue Gram stain, detects only a small portion of the organisms that are observed by immunofluorescence(Walker, 1989).
   7. Immunostaining :
      1. Time to Perform: unknown
      2. Description: Another approach to Rocky Mountain spotted fever diagnostics is immunostaining. This method is used by taking a skin biopsy of the rash from an infected patient prior to therapy or within the first 48 hours after antibiotic therapy has been started. Because rickettsiae are focally distributed in lesions of Rocky Mountain spotted fever, this test may not always detect the agent. Even in laboratories with expertise in performing this test, the sensitivity is only about 70% on biopsied tissues. This assay may also be used to test tissues obtained at autopsy and has been used to confirm Rocky Mountain spotted fever in otherwise unexplained deaths. Immunostaining for spotted fever group rickettsiae is offered by the CDC, a few state health departments, and some university-based hospitals and commercial laboratories in the United States(Website 4).
      3. False Negative: 30%(Website 4).
      4. Picture(s):
         • Immunostaining (Website 4)
           
           
           
           Description: Red structures indicate immunohistological staining of Rickettsia rickettsii in endothelial cells of a blood vessel from a patient with fatal RMSF. Copyright: CDC(Website 4).
   8. Microimmunofluorescence test :
      1. Time to Perform: unknown
      2. Description: A microimmunofluorescence test was used to study antibody responses to various spotted fever group and typhus group rickettsiae during Rocky Mountain spotted fever (RMSF) and epidemic typhus (ET). Patients with RMSF reacted most strongly to Rickettsia rickettsii; those with ET reacted predominantly to R. prowazekii. The degree of cross-reaction to other rickettsial strains varied from patient to patient, but a particular pattern of cross-reaction was consistently observed in serial sera from the same patient(Philip et al., 1976).
      3. False Positive: Cross-reactions of varying degree sometimes occurred to antigens both within and between the spotted fever and typhus group(Philip et al., 1976).
2. Immunoassay Test:
   1. Weil-Felix test :
      1. Time to Perform: unknown
      2. Description: The Weil-Felix test is based on the detection of antibodies to various Proteus species which contain antigens with cross-reacting epitopes to antigens from members of the genus Rickettsia with the exception of R. akari. Whole cells to Proteus vulgaris OX-2 react strongly with sera from persons infected with SFG rickettsia with the exception of those with RMSF, and whole cells of P. vulgaris OX-19 react with sera from persons infected with typhus group rickettsiae as well as with RMSF(La Scola and Raoult, 1997). By the Weil-Felix test, agglutinating antibodies are detectable after 5 to 10 days following the onset of symptoms, with the antibodies detected being mainly of the immunoglobulin M (IgM) type(La Scola and Raoult, 1997). The poor sensitivity and specificity of the Weil-Felix test are now well demonstrated for the diagnosis of RMSF, MSF, murine typhus, epidemic typhus, and scrub typhus. Although a good correlation between the results of the Weil-Felix test and detection of IgM antibodies by an immunofluorescence assay is often observed, with the development of techniques that are used to grow rickettsiae, this test should be used only as a first line of testing in rudimentary hospital laboratories(La Scola and Raoult, 1997). In most hospitals the laboratory diagnosis of RMSF is synonymous with the archaic, nonspecific, insensitive Weil-Felix test. Early in this century, the agglutination of certain strains of Proteus vulgaris by sera of patients convalescent from typhus fever was recognized. This phenomenon depends on antigens shared by P. vulgaris OX-19 and OX-2 and R. prowazekii, R. typhi, R. rickettsii, R. conorii, R. sibirica, and R. australis. Between 5 and 12 days after onset os symptoms, antibodies appear that agglutinate P. vulgaris OX-19 in 70% of patients and agglutinate P. vulgaris OX-2 in 47%. In addition to this poor level of sensitivity, another drawback is lack of specificity. Many healthy persons have agglutinating antibodies to P. vulgaris OX-19(Walker, 1995).
   2. Latex Agglutination :
      1. Time to Perform: unknown
      2. Description: Both indirect hemagglutination assay (IHA) and latex agglutination rely on a common source of rickettsial antigen, a protein-carbohydrate complex extracted from R. rickettsii by heat and alkaline conditions. This antigenic material is coated onto sheep or human type O erythrocytes for IHA and onto latex beads for latex agglutination. Serologic reactivity and binding to the erythrocytes require carbohydrate constituents, and trypsin digestion of the protein constituents does not affect the reaction of the antigen with antibody. Even though IHA demonstrated the earliest, steepest rise in antibody titer of all serologic tests for RMSF, it is seldom diagnostic in the acute stage of illness. The median antibody titer during days 4 to 16 of illness, when antirickettsial treatment must be initiated, is 16. Only 19% of patients with RMSF had an acute titer of 40, a lower value than the CCD criterion for the single titer indicating a probable diagnosis (greater than or equal to 128). The latex agglutination test is technically simple and rapid and requires no elaborate equipment. Endpoint determination seems to be difficult for some technologists. Antibodies to R. rickettsii are detcted 7 to 9 days after onset of illness and fall to nondiagnostic titers within 2 months. Perisitently detectable antibodies by IFA make this test appropriate for study of the prevalence of antibodies. The latex agglutination test is inappropriate for serosurveys and is more diagnostically discriminaotry for establishing the diagnosis of a recent infection. It is quite logical that hospital laboratories should replace the insensitive nonspecific Weil-Felix test with the commercially available latex agglutination test(Walker, 1989). The most imporant concept regarding serologic diagnosis is the emphatic insistence that it must be regarded as a retrospective confirmation of the clinical diagnosis. Currently, available serologic methods should not be considered as rapid acute diagnostic tests. Very seldom are specific antibodies to R. rickettsii detected during the acute stage of illness when empiric treatment must begin(Walker, 1989).
   3. Indirect Hemagglutination Assay :
      1. Time to Perform: unknown
      2. Description: Both indirect hemagglutination assay (IHA) and latex agglutination rely on a common source of rickettsial antigen, a protein-carbohydrate complex extracted from R. rickettsii by heat and alkaline conditions. This antigenic material is coated onto sheep or human type O erythrocytes for IHA and onto latex beads for latex agglutination. Serologic reactivity and binding to the erythrocytes require carbohydrate constituents, and trypsin digestion of the protein constituents does not affect the reaction of the antigen with antibody. Even though IHA demonstrated the earliest, steepest rise in antibody titer of all serologic tests for RMSF, it is seldom diagnostic in the acute stage of illness. The median antibody titer during days 4 to 16 of illness, when antirickettsial treatment must be initiated, is 16. Only 19% of patients with RMSF had an acute titer of 40, a lower value than the CCD criterion for the single titer indicating a probable diagnosis (greater than or equal to 128)(Walker, 1989). The most important concept regarding serologic diagnosis is the emphatic insistence that it must be regarded as a retrospective confirmation of the clinical diagnosis. Currently, available serologic methods should not be considered as rapid acute diagnostic tests. Very seldom are specific antibodies to R. rickettsii detected during the acute stage of illness when empiric treatment must begin(Walker, 1989).
   4. Agglutination by Convalsecent Serum :
      1. Time to Perform: unknown
      2. Description: Agglutination of convalsecent serum of microorganisms that are the etiologic agents of an infection was one of the earliest assays for antibodies to antigens of the microorganism itself. Unfortunately, it is virtually impossible to produce sufficient quantites of purified rickettsiae to use diagnostic agglutination tests routinely even with micromethods. Cultivation and purification of large concentrations of R. rickettsii require particular care to avoid inhalation of infectious aerosols. In addition, these pathogenic organisms destroy the host cells in which they are cultivated before a substaintial quantity of rickettsiae have accumulated, and separation of these oligate, intracellular bacteria from host cell components is quite inefficient. It is unreasonable to believe that R. rickettsii will ever be commercially available for the microaggluutination assay. Although the assay is specific, the sensitivity has varied according to the methods of purifiction of the rickettsiae. At a titer of greater than or equal to 32, the sensitivity of the assay for diagnosis of RMSF was reported as 56%(Walker, 1989).
      3. False Negative: 44%(Walker, 1989).
   5. Complement Fixation :
      1. Time to Perform: unknown
      2. Description: For a generation, the complement fixation test, using a soluble mixture of ether-extracted protein and LPS antigens of R. rickettsii, R. conorii, R. sibirica, R. akari, and R. australis. Washed rickettsial antibodies to species-specific antigens may be used to detect antibodies to species-specific antigens, but more effort is required to produce them. The complement fixation test is highly reproducible and specific. False-positive results are rare at a serum dilution of 1:16. Unfortunately, the sensitivity of the test is poor, because complement fixation antibodies are detected late in convalescence and in relatively small proportion of patients. Another drawback is that 5 to 10% of sera are anticomplementary. That is, they fix complement even in the absence of rickettsial antigen. The complement fixation test is rarely performed in the United States at present. It has become mainly a subject of historic concern because the major producer of rickettsial antigen for the assay, the CDC, recently ceased supplying the antigen to laboratories in this country(Walker, 1989). The most important concept regarding serologic diagnosis is the emphatic insistence that it must be regarded as a retrospective confirmation of the clinical diagnosis. Currently, available serologic methods should not be considered as rapid acute diagnostic tests. Very seldom are specific antibodies to R. rickettsii detected during the acute stage of illness when empiric treatment must begin(Walker, 1989).
      3. False Positive: False-positive results are rare at a serum dilution of 1:16(Walker, 1989).
3. Nucleic Acid Detection Test: