Experimental Scrub Typhus Research Articles

Strong interferon-gamma mediated cellular immunity to scrub typhus demonstrated using a novel whole cell antigen ELISpot assay in rhesus macaques and humans.

Scrub typhus is a febrile infection caused by the obligate intracellular bacterium Orientia tsutsugamushi, which causes significant morbidity and mortality across the Asia-Pacific region. The control of this vector-borne disease is challenging due to humans being dead-end hosts, vertical maintenance of the pathogen in the vector itself, and a potentially large rodent reservoir of unclear significance, coupled with a lack of accurate diagnostic tests. Development of an effective vaccine is highly desirable. This however requires better characterization of the natural immune response of this neglected but important disease. Here we implement a novel IFN-γ ELISpot assay as a tool for studying O. tsutsugamushi induced cellular immune responses in an experimental scrub typhus rhesus macaque model and human populations. Whole cell antigen for O. tsutsugamushi (OT-WCA) was prepared by heat inactivation of Karp-strain bacteria. Rhesus macaques were infected intradermally with O. tsutsugamushi. Freshly isolated peripheral blood mononuclear cells (PBMC) from infected (n = 10) and uninfected animals (n = 5) were stimulated with OT-WCA, and IFN-γ secreting cells quantitated by ELISpot assay at five time points over 28 days. PBMC were then assayed from people in a scrub typhus-endemic region of Thailand (n = 105) and responses compared to those from a partially exposed population in a non-endemic region (n = 14), and to a naïve population in UK (n = 12). Mean results at Day 0 prior to O. tsutsugamushi infection were 12 (95% CI 0–25) and 15 (2–27) spot-forming cells (SFC)/106 PBMC for infected and control macaques respectively. Strong O. tsutsugamushi-specific IFN-γ responses were seen post infection, with ELISpot responses 20-fold higher than baseline at Day 7 (mean 235, 95% CI 200–270 SFC/106 PBMC), 105-fold higher at Day 14 (mean 1261, 95% CI 1,097–1,425 SFC/106 PBMC), 125-fold higher at Day 21 (mean 1,498, 95% CI 1,496–1,500 SFC/106 PBMC) and 118-fold higher at Day 28 (mean 1,416, 95% CI 1,306–1,527 SFC/106 PBMC). No significant change was found in the control group at any time point compared to baseline. Humans from a scrub typhus endemic region of Thailand had mean responses of 189 (95% CI 88–290) SFC/106 PBMC compared to mean responses of 40 (95% CI 9–71) SFC/106 PBMC in people from a non-endemic region and 3 (95% CI 0–7) SFC/106 PBMC in naïve controls. In summary, this highly sensitive assay will enable field immunogenicity studies and further characterization of the host response to O. tsutsugamushi, and provides a link between human and animal models to accelerate vaccine development.

Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus.

Scrub typhus is a potentially lethal infection that is caused by the obligate intracellular bacterium Orientia tsutsugamushi The roles of Toll-like receptor 2 (TLR2) and TLR4 in innate recognition of O. tsutsugamushi have not been elucidated. By overexpression of TLR2 or TLR4 in HEK293 cells, we demonstrated that TLR2, but not TLR4, recognizes heat-stable compounds of O. tsutsugamushi that were sensitive to treatment with sodium hydroxide, hydrogen peroxide, and proteinase K. TLR2 was required for the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by dendritic cells. In an intradermal mouse infection model, TLR2-deficient mice did not show impaired control of bacterial growth or reduced survival. Moreover, after intraperitoneal infection, TLR2-deficient mice were even more resistant to lethal infection than C57BL/6 wild-type mice, which showed stronger symptoms and lower survival rates during the convalescent phase. Compared to the time of reduction of bacterial loads in TLR2-deficient mice, the reduction of bacterial loads in infected organs was accelerated in wild-type mice. The higher mortality of wild-type mice was associated with increased concentrations of serum alkaline phosphatase but not aspartate aminotransferase. The transcription of mRNA for TNF-α and IL-6 decreased more rapidly in peritoneum samples from wild-type mice than in those from TLR2-deficient mice and was therefore not a correlate of increased susceptibility. Thus, although TLR2 is an important mediator of the early inflammatory response, it is dispensable for protective immunity against O. tsutsugamushi Increased susceptibility to O. tsutsugamushi infection in TLR2-competent mice rather suggests a TLR2-related immunopathologic effect.

Dissemination of Orientia tsutsugamushi and inflammatory responses in a murine model of scrub typhus.

Central aspects in the pathogenesis of scrub typhus, an infection caused by Orientia (O.) tsutsugamushi, have remained obscure. Its organ and cellular tropism are poorly understood.The purpose of this study was to analyze the kinetics of bacterial dissemination and associated inflammatory responses in infected tissues in an experimental scrub typhus mouse model, following infection with the human pathogenic strain Karp. We provide a thorough analysis of O. tsutsugamushi infection in inbred Balb/c mice using footpad inoculation, which is close to the natural way of infection. By a novel, highly sensitive qPCR targeting the multi copy traD genes, we quantitatively monitored the spread of O. tsutsugamushi Karp from the skin inoculation site via the regional lymph node to the internal target organs. The highest bacterial loads were measured in the lung. Using confocal imaging, we also detected O. tsutsugamushi at the single cell level in the lung and found a predominant macrophage rather than endothelial localization. Immunohistochemical analysis of infiltrates in lung and brain revealed differently composed lesions with specific localizations: iNOS-expressing macrophages were frequent in infiltrative parenchymal noduli, but uncommon in perivascular lesions within these organs. Quantitative analysis of the macrophage response by immunohistochemistry in liver, heart, lung and brain demonstrated an early onset of macrophage activation in the liver. Serum levels of interferon (IFN)-γ were increased during the acute infection, and we showed that IFN-γ contributed to iNOS-dependent bacterial growth control.Our data show that upon inoculation to the skin, O. tsutsugamushi spreads systemically to a large number of organs and gives rise to organ-specific inflammation patterns. The findings suggest an essential role for the lung in the pathogenesis of scrub typhus. The model will allow detailed studies on host-pathogen interaction and provide further insight into the pathogenesis of O. tsutsugamushi infection.

恙虫病の感染免疫の研究

The immune response in human tsutsugamushi disease (scrub typhus) was studied. Anti-rickettsial activity of sera, peripheral mononuclear cells and their culture supernatants from patients on in vitro growth of Rickettsia tsutsugamushi proliferating in normal human peripheral macrophages was examined. The results obtained were as follows. 1) Sera from patients at the early convalescent stage, which exhibited high antibody titers against R. tsutsugamushi, effectively inhibited their growth in macrophages. 2) Sera from patients after a long period from the onset showed low antibody titers and did not inhibit rickettsial growth. 3) Mononuclear cells and T cell enriched fractions suppressed rickettsial growth when they were obtained from the patients at the early convalescent stage and even after as long as 3.5 years from the onset. 4) The culture supernatants of the T cell-enriched fractions which were collected from the patients mentioned above, exhibited a similar antirickettsial activity. These findings indicate that sensitized T lymphocytes and macrophages might play a fundamental role in immunological defense mechanism in tsutsugamushi disease. And the results obtained in our experiments are compatible with those previously reported in experimental scrub typhus in laboratory animals such as mice and monkeys.

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.

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.

Host defenses in experimental scrub typhus: inflammatory response of congenic C3H mice differing at the Ric gene.

Two strains of C3H mice differed in their susceptibility to lethal infection with Rickettsia tsutsugamushi strain Gilliam. Adult C3H/RV mice were markedly more resistant to lethal infection than C3H/HeDub mice, and both were histocompatible as assessed by mixed-lymphocyte cultures and graft-versus-host responses. The inflammatory response of susceptible C3H/HeDub mice to intraperitoneal infection was evident approximately 5 days postinfection, and the magnitude of the cellular influx increased until death of the animal. The inflammation consisted of an early polymorphonuclear leukocyte response, followed by a mononuclear cell influx which persisted until death of the animal. The C3H/RV mice evidenced similar kinetics of cell influx, but the inflammatory response was significantly reduced in magnitude, and the response of C3H/RV animals to Gilliam was predominantly mononuclear in nature, with little influx of polymorphonuclear leukocytes into the peritoneal cavity. C3H/RV mice were rendered susceptible to Gilliam infection by induction of a nonspecific inflammation with thioglycolate if given 3 days after infection. Conversely, treatment of C3H/HeDub mice with indomethacin, an anti-inflammatory agent, prolonged survival after infection with Gilliam. The results of this study indicate that genetic resistance to Gilliam is not due simply to a greater host response to infection or, conversely, that susceptibility is due to a host response quantitatively lacking in a cellular component necessary for antirickettsial immunity.

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.

Host defenses in experimental scrub typhus: mapping the gene that controls natural resistance in mice.

Natural resistance of mice to lethal ifections of Rickettsia tsutsugamushi, strain Gilliam, is controlled by a single, autosomal, dominant gene, which we have designated Ric, with r and s representing the resistant nd susceptible alleles, respectively. Using three sets of recombinant inbred mouse strains (BXD, BXH, and BXJ), the Ric locus was mapped to Chromosome 5 closely linked to the retinal degeneration (rd) locus. This linkage was confirmed by a backcross analysis. Based on the RI strains and the C57BL/6Ty-le congenic strain (the only proven Ric-rd cross-over), we estimate the recombination frequency between Ric and rd to be 0.015. Three presumptive Ric-rd recombinants detected among 93 backcross mice may represent caes of incomplete penetrance of the resistance allele rather than recombination. Analyis of th C57BL/6JTy-le congenic strain indicates that Ric is proximal to rd on Chromosome 5. If so, the correct gene order is Pgm-1-W-Ric-rd-Gus.

Host Defenses in Experimental Scrub Typhus: Role of Normal and Activated Macrophages

Host Defenses in Experimental Scrub Typhus: Role of Normal and Activated Macrophages

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: 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.

Host defenses in experimental scrub typhus: effect of chloramphenicol

The effect of chloramphenicol treatment on the development of immunity to scrub typhus in mice was studied. Chemotherapy was administered either shortly before infection and for 14 days thereafter (group I), or from 7 to 21 days postinfection (group II). Although the full course of either regimen resulted in complete protection of the mice against subsequent challenge with the homologous strain of Rickettsia tsutsugamushi, initiation of chemotherapy at 7 days postinfection resulted in more rapid development of immunity against both the original infection and subsequent challenge. In both treatment groups, a 1- to 2- day hiatus was observed between immunity to challenge in the treated animal and the ability to transfer this immunity to syngeneic recipients with lymphocyte-enriched spleen cells. Similarly, complement-fixing antibodies were not detectable until shortly after the animals were able to resist challenge. These data supported the conclusion that the rickettsiostatic effect of chloramphenicol allows the infected animal time to mount an effective immune response and, further, that initiation of chemotherapy early in the infection may delay development of this response.

Host Defenses in Experimental Scrub Typhus: Role of Spleen and Peritoneal Exudate Lymphocytes in Cellular Immunity

Lymphocytes obtained from spleens or peritoneal exudates of immune donor mice were evaluated for their ability to passively confer protection on recipients subsequently challenged with virulent scrub typhus rickettsiae. Peritoneal exudate lymphocytes (PELs) injected intraperitoneally were able to transfer complete protection against rickettsial challenge by 5 days after immunization, whereas splenic lymphocytes (SpL's) required 15 days to exhibit similar resistance. When immune lymphocytes were transferred intravenously, cells from both anatomical compartments required 15 days after immunization before they were able to completely protect recipients. PELs maintained this protective capacity for 2 weeks, but the passive immunity induced by intravenously transferred SpL's rapidly diminished to insignificant levels. It was particularly interesting that the protective effect of SpL's could be dramatically reduced by the concomitant presence of a mineral oil-induced peritoneal exudate. Almost total abrogation of resistance was observed when SpL's obtained from exudate-bearing mice were transferred intravenously. The protective capacity of both PELs and SpL's was resistant to 1,200 rads of gamma radiation at 7 to 10 days after immunization, but resistance was transient and by 3 weeks was undetectable. It was not possible to determine from this study whether the transferred lymphocytes were proximate mediators of protection in scrub typhus infection of mice or whether they served to recruit the host's own defenses, or both. However, it was possible to conclude that PEL's and SpLs exhibited functional heterogeneity and that PELs were more efficient mediators of protection.

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.