Gamma-irradiated scrub typhus immunogens: development of cell-mediated immunity after vaccination of inbred mice.

Mice immunized with three injections of gamma-irradiated Karp strain of Rickettsia tsutsugamushi were evaluated for the presence of cell-mediated immunity by using delayed-type hypersensitivity, antigen-induced lymphocyte proliferation, and antigen-induced lymphokine production. These animals also were evaluated for levels of circulating antibody after immunization as well as for the presence of rickettsemia after intraperitoneal challenge with viable Karp rickettsiae. After immunization with irradiated Karp rickettsiae, a demonstrable cell-mediated immunity was present as evidenced by delayed-type hypersensitivity responsiveness, lymphocyte proliferation, and production of migration inhibition factor and interferon by immune spleen lymphocytes. Also, a reduction in circulating rickettsiae was seen in mice immunized with irradiated rickettsiae after challenge with 1,000 50% mouse lethal doses of viable, homologous rickettsiae. All responses except antibody titer and reduction of rickettsemia were similar to the responses noted in mice immunized with viable organisms. Antibody levels were lower in mice immunized with irradiated rickettsiae than in mice immunized with viable rickettsiae. Furthermore, mice that were immunized with viable rickettsiae demonstrated markedly lower levels of rickettsemia after intraperitoneal challenge compared with either mice immunized with irradiated rickettsiae or nonimmunized mice.     

Development of specific and cross-reactive lymphocyte proliferative responses during chronic immunizing infections with Rickettsia tsutsugamushi

The development of antigen-responsive lymphocytes was followed in mice immunized with the Gilliam, Karp, or Kato strains of Rickettsia tsutsugamushi by utilizing an in vitro lymphocyte proliferation assay. Subcutaneous immunization with viable rickettsiae of all three strains resulted in the appearance of lymphocytes in the spleen responding to irradiated tissue culture-grown rickettsiae used as stimulating antigens. Although all animals demonstrated antigen-induced proliferation elicited by homologous antigen by 14 days after immunization, the time of peak responsiveness varied, depending on the strain of rickettsiae used for immunization. In all cases, peak proliferative responses occurred at a time after immunization that was after the previously reported time after immunization at which resistance to rechallenge was observed. Reactivity to heterologous strains of R tsutsugamushi developed roughly in parallel with homologous reactivity in Karp- and Gilliam-immunized mice, with a marked degree of heterologous reactivity evident. Kato-immunized mice demonstrated greater reactivity to heterologous antigens early in the development of antigen reactivity and demonstrated a somewhat greater degree of cross-reactivity, relative to homologous responses, than the other groups. It was found that nylon wool-nonadherent immune cells, if cultured with antigen and adherent cells obtained from normal spleens or peritoneal exudates, responded in culture. The thymus-derived lymphocyte nature of the responding cell was further suggested when treatment of immune spleen cells with anti-Thy 1.2 serum and complement eliminated antigen response.     

Gamma-irradiated scrub typhus immunogens: broad-spectrum immunity with combinations of rickettsial strains.

Scrub typhus immunogens were prepared from Rickettsia tsutsugamushi strains Karp, Kato, Gilliam, Kostival, and Buie by exposing frozen infected yolk sac suspensions to 300 krad of gamma radiation. Mouse protection tests showed that each of the irradiated immunogens protected C3H/HeDub mice against high challenge levels of Karp and Gilliam, but that none of these single-strain immunogens were capable of protecting against all five of the challenge strains. Broad-spectrum protection was achieved by using combinations of three strains of irradiated rickettsiae in a vaccination regimen of three injections at 5-day intervals. A comparison of vaccination efficacy employing three such combinations (Karp-Gilliam-Kato, Karp-Kostival-Kato, and Buie-Kostival-Kato) indicated that both sequential administration of strains on successive vaccination days and multiple injections of trivalent mixtures produced protective responses superior to those obtained with single-strain immunogens. Trivalent mixtures of rickettsiae exhibited a striking synergistic effect on the immune response of C3H/HeDub mice and elicited a protective response against Kato challenge that could not be obtained with any single-strain immunogen. Mice vaccinated with the trivalent Karp-Gilliam-Kato immunogen resisted challenge with more than 10(3) 50% mouse lethal doses of Karp and Gilliam for 12 months, and were resistant to similar levels of challenge with Kato and Buie for 6 months.     

Gamma interferon production in response to homologous and heterologous strain antigens in mice chronically infected with Rickettsia tsutsugamushi.

The ability of antigen-responsive, thymus-derived lymphocytes to produce immune (gamma) interferon was investigated during the development and expression of cellular immunity to Rickettsia tsutsugamushi. C3H/HeDub mice infected subcutaneously with the Gilliam strain developed the ability to produce serum interferon in response to intravenously inoculated antigen which correlated with the development of resistance to intraperitoneal rechallenge. Antigen-responsive lymphocytes, measured by interferon production and proliferation, were first apparent in draining lymph node cells, but spleen cell responses were detectable relatively soon after the appearance of reactive lymph node cells. The peak spleen cell response was of a greater magnitude and was found to be relatively long-lived. Reactivity to heterologous strains of R. tsutsugamushi also developed after immunization and paralleled the homologous responses, although reactivity was greatest to homologous antigens. Responses to heterologous strains differed in magnitude and time of appearances; however, immune mice resisted challenge with all strains of R. tsutsugamushi tested.     

Host defenses in experimental scrub typhus: role of normal and activated macrophages.

Resident peritoneal macrophage from BALB/c mice were infected in vitro with Rickettsia tsutsugamushi strain Gilliam, and rickettsial growth was estimated by microscopic examination of Giemsa-stained cells. Both number of infected macrophage per culture and number of intracellular rickettsiae per cell increased with time during culture. Treatment of rickettsiae with immune serum before infection macrophage cultures reduced the number of infected macrophage by 50%. Macrophage treated in vitro with lymphokines were able to suppress rickettsial growth in the absence of detectable antibody and exhibited a 75% reduction in infection compared with normal macrophage. We also obtained activated macrophage from immune mice and found that they were refractory to in vitro rickettsial infection. Macrophage populations activated in vitro or in vivo contained a small percentage of cells which supported unrestrained growth of rickettsiae. These data suggest that an early immunological event in experimental scrub typhus infection may be the development of activated macrophage capable of suppressing rickettsial proliferation before the appearance of circulating antibody.     

Host defenses in experimental scrub typhus: role of cellular immunity in heterologous protection.

The relative contributions of cellular and humoral immunity in scrub typhus infections were studied in inbred mice employing paired strains of Rickettsia tsutsugamushi differing in virulence. An infectious dose (100 MID50) of the less virulent Gilliam strain resulted in heterologous immune protection against an otherwise lethal challenge (1,000 MLD50) of the virulent Karp strain. Partial heterologous protection against lethal Karp challenge was observed in animals preimmunized with the Gilliam strain as early as 3 days prior to challenge, whereas complete protection against illness and death existed in animals immunized at least 7 days prior to challenge. In the heterologous protection provided by prior Gilliam infection, the role of humoral immunity was not of primary importance for the following reasons: (i) significant levels of complement-fixing antibody against R. tsutsugamushi were not detectable until long after animals were solidly immune; (ii) antibody eventually appearing after Gilliam immunization exhibited a consistently low complement-fixing titer against the immunizing homologous (Gilliam) strain and contained no detectable activity against the heterologous challenge (Karp) strain; and (iii) passive transfer of large quantities of serum from Gilliam immune mice, themselves immune to Karp challenge, failed to protect recipients against a similar challenge. However, protection was afforded by the passive transfer of serum containing antibody against Karp, suggesting a major role for antibody in protection against homologous infection. This heterologous challenge system was particularly useful because it minimized the role of humoral immunity, at least early in the course of infection, and allowed a definitive examination of the cellular response. Cell-mediated immunity played a major role in the heterologous protection observed after Gilliam immunization. This was evidenced by the significant protection against Karp challenge afforded by the passive transfer of spleen cells from animals immunized with Gilliam 7 to 63 days previously. Of the immune spleen cells, only those which were nonadherent, presumably lymphocytes, were capable of transferring passive heterologous protection. This protective effect of nonadherent cells could be ablated by depleting the cell population of thymus-derived or T cells with anti-theta serum and complement prior to transfer but not by use of anti-immunoglobulin serum and complement, which selectively removes bone marrow-derived or B cells. These results suggested that the cell in immune spleens capable of conferring heterologous protection was a T lymphocyte.     

Establishment and characterization of a T-cell line specific for Rickettsia tsutsugamushi.

To study the immunological protective system against rickettsial infection, a T-cell line specific for Rickettsia tsutsugamushi antigen was established by long-term culture of splenocytes from mice immunized with live Gilliam strain R. tsutsugamushi and then propagated in the presence of homologous rickettsial antigen and syngeneic filler cells. The characteristics of the T-cell line and its capacity to induce antirickettsial protection in vivo were studied. Flow cytometric analysis demonstrated that the T-cell line showed the phenotype Thy-1.2+ L3T4+ Lyt-2-, suggestive of helper T cells. In a lymphocyte proliferation assay, this cell line showed a specific response to Gilliam antigen, partial cross-reactivity to Karp antigen, but no response to Kato antigen. The proliferative response of this T-cell line was filler cell dependent, and genetic restriction was observed between the T-cell line and filler cells. The T-cell line produced gamma interferon, one of the macrophage-activating factors, in cultures with specific antigen and was able to adoptively mediate antirickettsial protection in vivo. The data presented here suggest that antigen-specific helper T cells play an important role in protection against rickettsial infection.     

Host Defenses in Experimental Scrub Typhus: Delayed-Type Hypersensitivity Responses of Inbred Mice

Delayed-type hypersensitivity responses of inbred mice during the course of lethal and chronic infections with strains of Rickettsia tsutsugamushi were evaluated by using the influx of radiolabeled cells into antigen-injected ears. Congenic strains of C3H mice, which previously have been shown to be resistant (C3H/RV) or sensitive (C3H/HeDub) to lethal intraperitoneal infection with the Gilliam strain of rickettsiae, both expressed delayed-type hypersensitivity early in the course of infection (5 to 7 days). The sensitive C3H/HeDub mice, however, exhibited a marked decline in reactivity just before death. In contrast, reactivity of C3H/RV mice remained high through day 9 and declined slowly through day 15 after infection. Similar results were obtained when BALB/c mice were infected with either the Karp or the Gilliam strain of rickettsiae, which produce a lethal or nonlethal infection, respectively, in this strain of mice. Rechallenge of C3H/RV mice elicited a rapid increase in reactivity, suggesting a secondary memory response. To analyze delayed-type hypersensitivity during chronic infection, C3H/HeDub mice were immunized by subcutaneous infection with the Gilliam strain of R. tsutsugamushi, and both delayed-type hypersensitivity reactivity and resistance to intraperitoneal challenge were examined. Delayed-type hypersensitivity reactivity developed slowly and peaked at 21 days postimmunization, which correlated with resistance to intraperitoneal challenge. Delayed-type hypersensitivity reactivity declined thereafter, but resistance to intraperitoneal challenge remained through 28 days postimmunization. Delayed-type hypersensitivity reactivity increased after secondary challenge at 28 days, again suggesting antigen memory generated by primary immunization. Transfer of delayed-type hypersensitivity reactivity was accomplished by using immune thymus-derived splenic lymphocytes isolated with nylon-wool columns. Abrogation of the ability of immune spleen cells to transfer delayed-type hypersensitivity reactivity after treatment with anti-Thy 1.2 alloantiserum and complement further supported the view that delayed-type hypersensitivity responses to scrub typhus rickettsiae were mediated by thymus-derived lymphocytes.     

Production of alpha and gamma interferons by spleen cells from cytomegalovirus-infected mice.

Antigen-induced lymphocyte proliferation and the production of a murine immune or type II interferon (MuIFN-gamma) by spleen cells in vitro were used to examine the cellular immune response to a cytomegalovirus infection of mice. Lymphocyte blastogenesis was induced by interaction with cytomegalovirus-infected mouse embryo fibroblasts in spleen cells from mice infected at least 6 days previously with cytomegalovirus. The peak of the blastogenic response occurred after 72 hr in culture with antigen. MuIFN was detected in cultures of cytomegalovirus-infected mouse embryo fibroblasts and spleen cells from both normal and infected mice. The MuIFN produced by spleen cells from normal or infected mice early during the course of the infection (days 1 to 2) was predominantly viral or type I interferon (MuIFN-alpha). Peak titers of MuIFN-alpha were present 24 to 48 h after exposure to antigen in vitro and before the peak of the blastogenic response. In contrast, spleen cells from mice infected at least 6 days previously produced both MuIFN-alpha and MuIFN-gamma in culture with the infected mouse fibroblasts. MuIFN-alpha was present early in the culture, before peak blastogenic activity. Peak levels of MuIFN-gamma were detected as lymphocyte blastogenic activity subsided. These results indicate that the cellular immune system of the murine host is capable of responding to cytomegalovirus infection, the afferent limb by antigen recognition and the efferent limb by the production of the lymphokine MuIFN-gamma.     

Macrophages in resistance to rickettsial infections: early host defense mechanisms in experimental scrub typhus.

Several early nonspecific host defense mechanisms were examined in resistant (BALB/c) and susceptible (C3H/He) mice after intraperitoneal inoculation with Rickettsia tsutsugamushi strain Gilliam. Inflammatory exudates were formed in both mouse strains in response to rickettsial inoculation, but the inflammatory response of C3H animals was delayed several days, and influx of peroxidase-positive macrophages occurred late in infection. Peritoneal cells of C3H mice became progressively infected, with 40% of both macrophages and lymphocytes containing intracellular rickettsiae by day 10. The early flammatory response of BALB/c mice was unexpectedly associated with a low percentage of infected peritoneal cells (1 to 2%). In vitro, no difference was detected in ability of resident macrophages of either strain to support the growth of R. tsutsugamushi or to become activated by treatment with lymphokines for rickettsiacidal activity. In vivo, however, macrophages from C3H mice inoculated with Gilliam were not activated on days 6 and 7 after infection, whereas BALB/c macrophages were continuously activated beginning on day 4. The lack of in vivo C3H macrophage activation was not secondary to deficient lymphokine production by infected lymphocytes, as levels of lymphokines produced by peritoneal lymphocytes of both strains were similar and peaked on day 7 after infection. Susceptibility to infection appears to be related to defective regulation of macrophage responses rather than to defects in macrophage function.     

Susceptibility of Rickettsia tsutsugamushi Gilliam to gamma interferon in cultured mouse cells.

Recombinant rodent gamma interferon (IFN-gamma) inhibited the infection of cultured BALB/3T3 mouse fibroblasts by Rickettsia tsutsugamushi Gilliam, apparently mainly by clearance of intracellular rickettsiae. No significant effect on rickettsial entry into the cells was noted; IFN-gamma was toxic to infected cells, as measured by the capacity of treated, infected cells to attach to the surfaces of culture vessels. In a small proportion of IFN-gamma-treated cells, rickettsial replication appeared to persist at normal levels. A fraction (28%) of rickettsiae clonally isolated from cultures treated with IFN-gamma was resistant to IFN-gamma-mediated inhibition, but four serial passages of these resistant clones in the absence of additional IFN-gamma resulted in the loss of resistance. In several respects, therefore, the IFN-gamma-mediated inhibition of scrub typhus rickettsiae in cultured fibroblasts was similar to that reported for Rickettsia prowazekii.     

Role of macrophages in innate and acquired host resistance to experimental scrub typhus infection of inbred mice.

Mechanisms of innate resistance to infection with the Gilliam strain of Rickettsia tsutsugamushi were examined using congenic strains of mice resistant (C3H/RV) or susceptible (C3H/He) to intraperitoneal infection. Both strains of mice were resistant to infection with 1,000 50% mouse lethal doses of rickettsiae if given intravenously. In both systems rickettsial replication occurred after intravenous infection, as evidenced by an increase in rickettsial numbers in the spleens of infected animals, followed by a decrease in rickettsiae to low levels by day 14 postinfection. Administration of the antimacrophage agents silica and carrageenan to C3H/He mice intravenously rendered these animals susceptible to lethal infection. Neither irradiation nor silica given individually rendered C3H/RV mice susceptible to intravenous infection. However, if silica and irradiation were given together, a lethal infection occurred after intravenous infection. C3H/RV mice became susceptible to lethal infection after sublethal doses of irradiation only if they were infected intraperitoneally. Administration of silica or carrageenan had no effect on the outcome of intraperitoneal infection of these mice with Gilliam rickettsiae. These data suggest that both strains of mice share innate resistance mechanisms to intravenous infection that consist of fixed macrophages. Resistance of C3H/RV mice to intraperitoneal infection, in contrast, apparently was dependent only on an irradiation-sensitive process.     

Effect of sublethal gamma radiation on host defenses in experimental scrub typhus.

The effect of sublethal gamma radiation on inbred mice chronically infected with scrub typhus rickettsiae was examined. Inbred mice which were inoculated with the Gilliam or Karp strain of Rickettsia tsutsugamushi by the subcutaneous route harbored the infection for at least 1 year. Irradiation of these animals at 12 or 52 weeks postinoculation with normally sublethal levels induced a significantly higher percentage of rickettsemic mice (recrudescence) than was seen in the unirradiated, similarly infected control animals. In addition, sublethal irradiation at 12 weeks induced a quantitative increase in total rickettsiae. Homologous antibody titers to the rickettsiae were examined for 5 weeks after irradiation to determine the role of the humoral response in radiation-induced recrudescence. Unirradiated, infected mice showed consistent titers of about 320 throughout the 5-week observation period, and the titer was not affected by exposure of up to 500 rads of gamma radiation. Drug dose-dependent radioprotection and modification of recrudescence was noted in infected, irradiated mice treated with the antiradiation compound S-2-(3-aminopropylamino)ethyl phosphorothioic acid. The results of this investigation supported the conclusion that the recrudescence of a chronic rickettsial infection in the appropriate host after immunological impairment due to gamma radiation can result in an acute, possibly lethal rickettsemia.     

Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice.

Pathogenicity of Rickettsia tsutsugamushi for laboratory mice is known to be influenced by at least three factors: (i) route of inoculation, (ii) antigenic strain, and (iii) natural resistance of the host. By using Karp, Gilliam, and Kato strains of R. tsutsugamushi, we examined the effect of these three pathogenicity factors on the kinetics of infection and the development of immunity in BALB/cDub and C3H/HeDub mice. The appearance of rickettsemia in the pathogenic infections generally preceded infections of reduced pathogenicity by 1 to 2 days in both magnitude and time of onset. Mice infected by the subcutaneous route with normally pathogenic rickettsiae, i.e., Gilliam-infected C3H/HeDub mice and Karp-infected BALB/cDub mice, consistently maintained a detectable rickettsemia over a 1-year period. Rickettsiae were recovered from the spleens of 95% (19 of 20) of these mice 52 weeks postinfection. In contrast, mice with infections of reduced pathogenicity, i.e., BALB/cDub mice infected by intraperitoneal and subcutaneous inoculation with Gilliam, did not have detectable rickettsemia from week 20 through week 52 postinfection except for a single mouse on week 44 postinfection. Rickettsiae were detected in the spleens of only 40% (8 of 20) of these mice after 1 year. In both Gilliam-infected mouse strains, protection against heterologous challenge with Karp or Kato rickettsial strains was incomplete up to 7 days postimmunization. Infections of reduced pathogenicity did not result from an enhanced systemic immune response by the host. The onset of the humoral response was not different for the pathogenic and reduced-pathogenicity infections. Pathogenicity differences seemed to result from the more rapid growth of the rickettsiae in the pathogenic infections.     

Antigens of scrub typhus rickettsiae: separation by polyacrylamide gel electrophoresis and identification by enzyme-linked immunosorbent assay.

Antigens of plaque-purified Rickettsia tsutsugamushi strains Gilliam, Karp, and Kato were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by an enzyme-linked immunosorbent assay. Six antigens were identified in each of the three prototype strains; in strain Gilliam, these antigens were located in the cell envelope fraction of the organisms. Reactivity of these isolated antigens with homologous or heterologous immune sera indicated that different macromolecules existed in all three strains, although they exhibited very similar mobilities during electrophoresis. Antigens of strain Gilliam reacted equally well with antibodies directed against Gilliam, Karp, or Kato rickettsiae. However, strains Karp and Kato each had two distinct antigens which did not react with heterologous antisera. R. tsutsugamushi antigens retained immunogenicity after electrophoresis, and antisera raised against them reacted with intact organisms and exhibited specificity in reactions with isolated antigens.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

Association of an inflammatory I region-associated antigen-positive macrophage influx and genetic resistance of inbred mice to Rickettsia tsutsugamushi

Strains of C3H mice differing in susceptibility to intraperitoneal infection with the Gilliam strain of Rickettsia tsutsugamushi were used to investigate the role of the I region-associated (Ia) antigen-bearing macrophage in the genetic resistance of mice to this organism. Resistant mice (C3H/RV) were found to produce a quantitatively greater Ia antigen-positive macrophage response after infection compared to mice (C3H/HeDub) which underwent a lethal infection. The macrophage influx produced in response to infection of the C3H/HeDub mice was deficient in Ia antigen-bearing cells, as evaluated by antigen presentation function and by the use of macrophages as stimulator cells in a mixed lymphocyte response. The resistance to infection, as well as the Ia-positive macrophage response in C3H/RV mice, was sensitive to 450 to 600 rads of irradiation. C3H/HeDub mice produced exudates rich in Ia-positive macrophages if stimulated with concanavalin A or after challenge with R. tsutsugamushi (if previously immunized), ruling out an innate inability of this strain of mice to produce Ia-positive macrophages exudates. Challenge of either strain of mice immunized by a prior subcutaneous infection resulted in a rapid (3 to 5 days) peak of Ia-positive macrophages responding to the peritoneal cavity. It also was noted that subcutaneous infection alone resulted in an increase in the proportion and number of "resident" macrophages which were Ia positive. These data suggest that the macrophage influx in terms of Ia-bearing cells is at least associated with the genetic resistance of C3H/RV mice to infection with this rickettsiae and may play a role in resistance. Furthermore, it would appear that the Ia-positive macrophage is a factor in acquired immunological resistance to reinfection.     

Plaque assay and cloning of scrub typhus rickettsiae in irradiated L-929 cells.

It was demonstrated that gamma-irradiated L-929 cells support plaque formation by three strains of Rickettsia tsutsugamushi and representative species of the spotted fever and typhus group rickettsiae. Sensitivity of the plaque assay for detection of viable scrub typhus rickettsiae was similar to that achieved with intraperitoneal inoculation of random-bred mice. The concentration of irradiated cells and the temperature and length of incubation were all found to affect plaque size. A technique combining terminal dilution and plaque purification was used to obtain clones of three strains of scrub typhus rickettsiae.     

Recombinant 56-kilodalton major outer membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity

The gene encoding the 56-kDa protein of Orientia tsutsugamushi Shanxi was amplified by a nested PCR and cloned into the expression vector pQE30. The 56-kDa protein of O. tsutsugamushi Shanxi (Sxh56) was expressed as a fusion protein with the His(6)-binding protein of Escherichia coli by deleting the signal peptide-encoding sequence from the 5' end of the open reading frame. The recombinant protein formed inclusion bodies when expressed in E. coli M15. The recombinant protein was examined for reactivity with mouse sera against three antigenic prototypes of O. tsutsugamushi by an immunoblot assay. The recombinant Sxh56 reacted only to polyclonal antiserum to O. tsutsugamushi Gilliam in an enzyme-linked immunosorbent assay (ELISA) and in an immunoblot assay. Recombinant Sxh56 was purified by Ni-nitrilotriacetic acid affinity chromatography and injected into mice to evaluate its ability to stimulate immune responses. High levels of immunoglobulin G and T-cell proliferation appeared in mice immunized with the recombinant protein. The recombinant Sxh56 was used in an ELISA to evaluate the ability of the method to detect antibodies to O. tsutsugamushi in human and animal sera. Thirty sera from mice infected with O. tsutsugamushi Gilliam or Shanxi and 55 sera from normal mice were detected in the ELISA with recombinant Sxh56, and the sensitivity and specificity were 96.67 and 100%, respectively. One hundred fifty-one positive sera and 412 negative sera to O. tsutsugamushi Gilliam were detected in an indirect immunofluorescence assay with the recombinant protein, and the sensitivity and specificity were 96.36 and 88.08%, respectively. These results strongly suggest that the recombinant Sxh56 is a suitable type-specific immunodiagnostic antigen and vaccine candidate.     

Host Defenses in Experimental Scrub Typhus: Genetics of Natural Resistance to Infection

Genetic resistance to lethal infection with Rickettsia tsutsugamushi was studied in over 30 inbred strains, inbred hybrids, and outbred stocks of mice. Inbred mice infected intraperitoneally with the Gilliam strain of R. tsutsugamushi showed three patterns of response: susceptible (A/HeJ, C3H/HeDub, C3H/HeJ, C3H/HeN, C3H/St, CBA/J, DBA/1J, DBA/2J, and SJL/J), resistant (AKR/J, BALB/cDub, BALB/cJ, C57BL/6J, C57L/J, and SWR/J), and selectively resistant (A/J). The selectively resistant pattern was characterized by random deaths occurring throughout the titration range and was also observed in three of the six outbred mouse stocks surveyed. No correlation was evident between the H-2 haplotype of inbred mice and their response to Gilliam infection. The progeny from five different Gilliam-resistant by Gilliam-susceptible inbred parental crosses were all resistant. Study of F(1), F(2), and parental backcross generations of BALB/cDub (resistant) and C3H/HeDub (susceptible) hybrids indicated resistance was dominant and was controlled by a single gene or a closely linked cluster of genes that were autosomal and not linked to coat color. The resistance of BALB/cDub mice was not due to an inability of host cells to support rickettsial growth, since C3H/HeDub and BALB/cDub embryo cell cultures supported similar growth of Gilliam organisms. C3H/HeDub mice, although susceptible to intraperitoneal Gilliam infection, were capable of mounting an immune response to Gilliam antigens, since subcutaneous infection was not lethal and did protect animals against subsequent intraperitoneal challenge with either the Gilliam or Karp strains of R. tsutsugamushi.