Experimental Rickettsia tsutsugamushi infections in dogs.

Dogs were infected intravenously and intradermally with the Gilliam and Karp strains of R. tsutsugamushi. Although the development of clinical signs was related to the dose of the organism, Gilliam-infected dogs developed severer infections than those infected with Karp. Specific antibodies were demonstrated in sera of experimentally infected dogs.     

Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.     

Assembly of Rickettsia tsutsugamushi progeny in irradiated L cells.

In the assembly of Rickettsia tsutsugamushi progeny in irradiated L cells, nascent forms first appear as undemarcated foci in the host cell granular cytoplasm, in which electron-lucent filamentous (f) and electron-dense granular (g) areas differentiate. Morphological observations indicated that the assembly involves formation of a filamentous network in the f area, manufacture of rickettsial ribosomes in the g area, and formation of mildly electron-dense fuzzy zones, along which a double membrane assembles.     

Growth pattern of Rickettsia tsutsugamushi in irradiated L cells.

Irradiated L cells infected with Rickettsia tsutsugamushi were studied under the electron microscope to define the morphological growth pattern of the organism. For 2 days after inoculation, no rickettsiae were found either extra- or intracellularly; after 2 days multiple rickettsiae appeared within the host cells without morphological evidence of their entry. These observations showed that the rickettsiae within the cell were assembled in situ by segregation of portions of the granular cytoplasm and subsequent internal differentiation and surface membrane assembly of the segregated bodies. The protoplasmic (P) bodies, which seemed to be formed by shedding infected-cell granular cytoplasm, consistently appeared on the surface and within the phagosomes of the host cells. Rickettsiae were occasionally seen entering host cells in the later phase of infection; these were apparently the ones assembled within the P bodies. This suggested that the P bodies, and not the rickettsiae, were the major infectious particles that transmitted the rickettsial genetic substance among the host cells. On the basis of the present morphological observations, viral-type multiplication for R. tsutsugamushi is proposed.     

Persistence and reactivation of Rickettsia tsutsugamushi infections in laboratory mice.

The persistence of R. tsutsugamushi in tissues of experimentally infected mice for 565 days was demonstrated. Reactivation of apparently dormant infections was accomplished by inoculating the mice with a heterologous strain of the organism or treatment with cyclophosphamide.     

Suppression of lymphocyte responsiveness during acute Rickettsia tsutsugamushi infection in mice

Lymphocytes from Inbred Balb/cyJ mice infected subcutaneously with 10(3) MLD50 of Rickettsia tsutsugamushi Karp strain were unresponsive to rickettsial antigens and phytohemagglutinin in the lymphocyte transformation assay between 11 and 28 days post-infection. There was, however, no correlation between this suppressed responsiveness to rickettsial antigens and the immune capacity to resist a normally lethal ip challenge with the Karp strain. Fourteen days after infection, these mice survived a potentially lethal Karp challenge. Naive recipients of day 14 post-infection mouse splenic lymphocytes also were partially protected against ip challenge. Karp strain-specific anti-rickettsial antibody reached a peak geometric mean titer of 260 during the period of apparent cellular unresponsiveness. However, day 14 post infection mice showed a fourfold decrease of antibody response to a T-dependent antigen, which suggests the presence of T-dependent antibody suppression in these mice.     

The effect of mouse age on the determination of Rickettsia tsutsugamushi virulence

The effect of age on the susceptibility of ICR mice to lethal intraperitoneal (ip), Rickettsia tsutsugamushi infections was tested with five virulent strains--Karp, Kato, Gilliam, TA763, and TH1817--and three strains of reduced virulence--TA678, TA686, and TA716. Susceptibility differences were noted only in the ICR mice inoculated with two of the strains of reduced virulence, TA716 and TA678. With both strains, mice in the 12-weeks and younger age groups had lower death rates than did mice in the 21-weeks and older age groups. Also, CBA/CaJ mice of varying ages were inoculated intravenously with large doses of the Gilliam strain to determine the effect of age on susceptibility to acute death syndrome (ADS). A progressive increase in ADS resistance was seen in the 4-, 8-, 12-, and 16-week-old age groups. This study indicates that the age of mice used to test the virulence of R. tsutsugamushi strains may be an important consideration, especially when testing the ip lethality of strains of reduced virulence.     

Electron Microscopic Studies on Intracellular Multiplication of Rickettsia tsutsugamushi in L Cells

The mechanism and kinetics of intracellular growth of Rickettsia tsutsugamushi were investigated by electron microscopic observations, parallel with quantitative analysis by counting the rickettsiae seen in electron micrographs and by plaque assay for infectivity of the culture. The observations demonstrated the existence of electron-less dense and -dense types of rickettsiae in the early stage of infection, binary fission and the process of release of the microorganisms in the host cell cytoplasm and from the cell surface, formation of abnormally long rickettsiae, and the process of lysis of the host cell in the later stage of infection with vacuole formation between the inner and outer leaflets of the host cell nuclear membrane. Separate titrations of infectivity of the cells and the culture fluid showed a very slow increase in infectivity in the culture fluid compared with the intracellular titer, suggesting that the progeny rickettsiae stay in the cell or at the cell surface for a relatively long period. Doubling time of the rickettsia was found to be about 9 hr.     

DNA base composition of Rickettsia tsutsugamushi determined by reversed-phase high-performance liquid chromatography

The DNA base composition of Rickettsia tsutsugamushi was determined by reversed-phase high-performance liquid chromatography and compared with that of Rickettsia rickettsii. The G+C contents were 28.1 to 30.5 mol% for R. tsutsugamushi and 32.1 mol% for R. rickettsii.     

Production and characterization of monoclonal strain-specific antibodies against prototype strains of Rickettsia tsutsugamushi

We have developed 18 hybridoma cell lines which secrete murine monoclonal strain-specific antibodies to prototype strains of Rickettsia tsutsugamushi: nine anti-Gilliam, four anti-Karp and five anti-Kato antibodies. All the monoclonal antibodies reacted only with their homologous strains in direct and indirect immunofluorescence (IF), or indirect immunoperoxidase (IP) test. By IF and IP tests with the monoclonal antibodies, 22 strains of R. tsutsugamushi, which were newly isolated from mites, field rodents and patients with Tsutsugamushi disease (scrub typhus) in Japan, were all clearly identified as either Gilliam or Karp type. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that the monoclonal antibodies recognized primarily the polypeptides of an apparent molecular weight of 54 to 56 kilodaltons of the homologous rickettsial surface. The monoclonal antibodies produced in the present study should enhance the serotyping and further analytical investigation of the rickettsial antigens since they recognize the strain- or type-specific polypeptides and do not show any cross-reaction among strains.