Inthawong et al 2024 Scrub typhus NHP model intradermal inoculation dose evaluation

Description

Scrub typhus, endemic to the Asia-Pacific region is caused by Orientia tsutsugamushi and transmitted to humans through bites of infected larval Leptotrombidium mites (chiggers). Currently, no protective vaccines exist. Animal models have substantially advanced our understanding of immune response dynamics, pathophysiology and pathogenesis of scrub typhus. Our group previously validated a non-human primate (NHP) model using an intradermal (ID) challenge approach. To further refine this model, we evaluated the effects of progressively increasing doses of human pathogenic O. tsutsugamushi on disease progression, clinical outcomes, bacterial kinetics and immune responses in rhesus macaques (Macaca mulatta). Macaques were inoculated ID with O. tsutsugamushi strain Karp at doses of 106, 107 and 107.8 based on murine median Lethal Dose (muLD50). All NHPs demonstrated characteristic signs of human scrub typhus, including eschar formation, regional lymphadenopathy, elevated body temperature, and bacteremia. Dose-dependent responses were observed, with higher doses (107 and 107.8 muLD50), showing more severe clinical signs and pronounced adaptive immune responses. Increased production of Th1 and Th2 cytokines, with a significant rise surrogate markers, were observed in the 107 muLD50 group. This study demonstrates the critical role of differential serum IFN- levels in shaping immunity, and suggests that the 107 muLD50 dose is optimal for reliably inducing surrogate markers and immune responses resembling human infection dynamics. Our study highlights the importance of thorough inoculum dose characterisation in animal models to ensure accurate and reproducible outcome measures. The fundamental findings will serve emerging research on scrub typhus, particularly in establishing experimental studies.

Steps to reproduce

Datasets or images for each figure are available in separate tabs within the excel files. Figure 1. Experimental scheme for this study. Figure 2. Eschar and lymphadenopathy development timecourse following ID inoculation. Figure 3. Body temperature and bacteremia. Heatmap of changes in body temperature compared to baseline, graph of bacteremia in O. tsutsugamushi (Ot)- or mock-inoculated rhesus macaques from 0-29 dpi and Spearman correlation between body temperature and bacteremia are shown for each O. tsutsugamushi inoculated groups. Figure 4. Kinetics of hematological and clinical chemistry profiles following ID inoculation. including total white blood cells (WBC), lymphocytes, neutrophils, and platelets, and C-reactive protein (CRP) levels in O. tsutsugamushi (Ot) - or mock-inoculated rhesus macaques from 0-28 dpi. Figure 5. Kinetics of cellular immune responses elicited by O. tsutsugamushi ID inoculation. The responses against HI-WCA-OT, 56 kDa and 47 kDa peptide megapools (mp) determined weekly by ex-vivo IFN-gamma ELISpot assay during the experimental interval of 28 days. Figure 6. Heatmap showing the relative differences in cytokine secretion kinetics. Fold change values within 2-fold of baseline were considered unchanged from baseline. n=5 for each group on days 7, 14, 21 and 28 dpi in mock controls and O. tsutsugamushi-inoculated rhesus macaques. . Figure 7. Relationships of bacteremia and host response parameters. Figure 8. Correlation matrix of bacteremia with host-responses following ID inoculation with O. tsutsugamushi and the involvement of IL-17 and IL-18 signaling pathway. S1 Fig. Representative images of eschar development at two left thigh inoculation sites in O. tsutsugamushi (Ot) or mock-inoculated rhesus macaques from 0-28 dpi. S2.1 Fig. Weekly kinetics of cytokine levels associated with Th1, Th2 and Th17 signaling pathways. S2.2 Fig. Weekly kinetics of chemokines and growth factors were monitored in rhesus macaques inoculated intradermally with O. tsutsugamushi (Ot) - or mock-inoculated rhesus macaques from 0-28 dpi. S1 Table. Observing and scoring of local inoculation site assessment by RISE SCORE S1.1 Table. RISE SCORE of Mock control S1.2 Table. RISE SCORE of 106 muLD50 group S1.3 Table. RISE SCORE of 107 muLD50 group S1.4 Table. RISE SCORE of 107.8 muLD50 group S2. Table. Minimal detection concentration of analytes from Milliplex kit S3.1 Table Complete blood counts and differential counts for mock control S3.2 Table. Complete blood counts and differential counts for 10E6 group S3.3 Table. Complete blood counts and differential counts for 10E7 muLD50 group S3.4 Table. Complete blood counts and differential counts for 10E7.8 muLD50 group S S4.2 Table. Blood chemistry parameters for 106 muLD50 group S4.1 Table. Blood chemistry parameters for mock control S4.3 Table. Blood chemistry parameters for 107 muLD50 group S4.4 Table. Blood chemistry parameters for 107.8 muLD50 group

Institutions

Categories

Funding

Licence