Characterization of the rhesus macaque (Macaca mulatta) scrub typhus model : Susceptibility to intradermal challenge with the human pathogen Orientia tsutsugamushi Karp

Background Scrub typhus is an important endemic disease in tropical Asia caused by Orientia tsutsugamushi for which no effective broadly protective vaccine is available. The successful evaluation of vaccine candidates requires well-characterized animal models and a better understanding of the immune response against O. tsutsugamushi. While many animal species have been used to study host immunity and vaccine responses in scrub typhus, only limited data exists in non-human primate (NHP) models. Methodology/Principle findings In this study we evaluated a NHP scrub typhus disease model based on intradermal inoculation of O. tsutsugamushi Karp strain in rhesus macaques (n = 7). After an intradermal inoculation with 106 murine LD50 of O. tsutsugamushi at the anterior thigh (n = 4) or mock inoculum (n = 3), a series of time course investigations involving hematological, biochemical, molecular and immunological assays were performed, until day 28, when tissues were collected for pathology and immunohistochemistry. In all NHPs with O. tsutsugamushi inoculation, but not with mock inoculation, the development of a classic eschar with central necrosis, regional lymphadenopathy, and elevation of body temperature was observed on days 7–21 post inoculation (pi); bacteremia was detected by qPCR on days 6–18 pi; and alteration of liver enzyme function and increase of white blood cells on day 14 pi. Immune assays demonstrated raised serum levels of soluble cell adhesion molecules, anti-O. tsutsugamushi-specific antibody responses (IgM and IgG) and pathogen-specific cell-mediated immune responses in inoculated macaques. The qPCR assays detected O. tsutsugamushi in eschar, spleen, draining and non-draining lymph nodes, and immuno-double staining demonstrated intracellular O. tsutsugamushi in antigen presenting cells of eschars and lymph nodes. Conclusions/Significance These data show the potential of using rhesus macaques as a scrub typhus model, for evaluation of correlates of protection in both natural and vaccine induced immunity, and support the evaluation of future vaccine candidates against scrub typhus.
Author summary Scrub typhus is a febrile illness caused by bacteria that invade and live within cells of the immune and blood vessel systems. Small earth-bound mites can bite humans and transmit these bacteria into the skin. Scrub typhus is treatable with antibiotics, but currently there is no scrub typhus vaccine available. Unfortunately if humans get scrub typhus, the immune response is usually weak and short-lived, especially against different strains, and affected individuals can get ill again within a year. This is a problem in areas where the infection is very common and a vaccine could be an effective approach to protect susceptible humans against scrub typhus. In this study, we characterized the immune response and disease features of scrub typhus in rhesus macaques by inoculating the bacteria directly into the skin–similar to the mite bite in nature–previously this had only been done in cynomolgus macaques (Macaca fascicularis). We found that the scrub typhus symptoms and immune responses of rhesus macaques resemble more closely the human responses than those of cynomolgus macaques. Studying the immune response in rhesus macaques will help us to understand how humans react against different bacterial proteins, to identify new markers of protection and to find the strongest vaccine candidates. This will then help us develop new and better vaccines (and also diagnostics) against scrub typhus in the future.