Orientia tsutsugamushi: An Unusual Intracellular Bacteria-Adaptation Strategies, Available Antibiotics, and Alternatives for Treatment.

During evolution Orientia tsutsugamushi became a smarter obligate bacterium to establish as intracellular pathogens. O. tsutsugamushi is a human pathogenic bacterium responsible for 1 billion infections of scrub typhus. Several novel mechanisms make this bacterium unique (cell wall, genetic constitutions, secretion system, etc.). In 2007, O. tsutsugamushi Boryong was pioneer strain for whole-genome sequencing. But the fundamental biology of this bacterial cell is a mystery till date. The unusual biology makes this organism as model for host cell interaction. Only a few antibiotics are effective against this intracellular pathogen but emergence of less susceptibility toward antibiotics make the situation alarming. The review was captivated to highlight the unusual aspects of adaptation, antibiotics, and drugs beyond antibiotics.

mNGS helped diagnose scrub typhus-associated HLH in children: a report of two cases.

Background: Scrub typhus, caused by the Orientia tsutsugamushi (Ot), is a widespread vector-borne disease transmitted by chigger mites. Hemophagocytic lymphohistiocytosis (HLH) is considered to be one of the potentially severe complications. The diagnosis of scrub typhus-associated HLH may be overlooked due to the non-specific clinical characteristics and the absence of pathognomonic eschar. Case presentation: We obtained clinical data from two patients in the South of Sichuan, China. The first case involved a 6-year-old girl who exhibited an unexplained fever and was initially diagnosed with sepsis, HLH, and pulmonary infection. The other patient presented a more severe condition characterized by multiple organ dysfunction and was initially diagnosed with septic shock, sepsis, HLH, acute kidney injury (AKI), and pulmonary infection. At first, a specific examination for scrub typhus was not performed due to the absence of a characteristic eschar. Conventional peripheral blood cultures yielded negative results in both patients, and neither of them responded to routine antibiotics. Fortunately, the causative pathogen Orientia tsutsugamushi (Ot) was detected in the plasma samples of both patients using metagenomics next-generation sequencing (mNGS) and further confirmed by polymerase chain reaction. Subsequently, they both were treated with doxycycline and recovered quickly. Conclusion: The unbiased mNGS provided a clinically actionable diagnosis for an uncommon pathogen-associated infectious disease that had previously evaded conventional diagnostic approaches.

Molecular epidemiological study of Scrub Typhus in residence, farm and forest habitats from Yunnan Province, China.

The number of people suffering from scrub typhus, which is not of concern, is increasing year by year, especially in Yunnan Province, China. From June 1, 2021 to August 15, 2022, a total of 505 mammalian samples were collected from farm, forest, and residential habitats with high incidence of scrub typhus in Yunnan, China, for nPCR (nested PCR) and qPCR (quantitative real-time PCR) detection of Orientia tsutsugamushi. A total of 4 orders of murine-like animals, Rodentia (87.52%, n = 442), Insectivora (10.29%, n = 52), Lagomorpha (1.79%, n = 9) and Scandentia (0.40%, n = 2) were trapped. Comparing the qPCR infection rates in the three habitats, it was no significant difference that the infection rate of residential habitat (44.44%) and that of the farm habitat (45.05%, P>0.05), which is much larger than that of the forest habitat (3.08%) (P<0.001). Three genotypes (Karp-like, Kato-like and TA763-like) of O. tsutsugamushi were found from Yunnan, China in this study.

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

BACKGROUND: Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. RESULTS: Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. CONCLUSIONS: P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Molecular detection of Orientia tsutsugamushi in ectoparasites & their small mammal hosts captured from scrub typhus endemic areas in Madurai district, India.

BACKGROUND OBJECTIVES: Scrub typhus, caused by Orientia tsutsugamushi present in small mammals harbouring the ectoparasites. A study was undertaken to detect the pathogen present in small mammals and its ectoparasites in the scrub typhus-reported areas. METHODS: The small mammals (rodents/shrews) and its ectoparasites were screened for O. tsutsugamushi using nested PCR amplification of the groEL gene. Small mammals were collected by trapping and screened for ectoparasites (mites, ticks and fleas) by combing method. RESULTS: All the chigger mites collected were tested negative for O. tsutsugamushi . Interestingly, adult non-trombiculid mites ( Oribatida sp., Dermanyssus gallinae ), fleas ( Xenopsylla astia, X. cheopis, Ctenophalides felis and Ctenophalides sp.) and ticks ( Rhipicephalus sanguineus , R. haemaphysaloides ) screened were found to be positive for O. tsutsugamushi , which the authors believe is the first report on these species globally. Bandicota bengalensis with O. tsutsugamushi infection is reported for the first time in India. The O. tsutsugamushi groEL sequences from the positive samples were similar to the reference strains, Karp and Ikeda and phylogenetically clustered in clade IV with less evolutionary divergence. The blood samples of Rattus rattus , Suncus murinus and B. bengalensis collected from this area were tested positive for O. tsutsugamushi ; interestingly, the sequence similarity was much pronounced with their ectoparasites indicating the transmission of the pathogen to host or vice versa . INTERPRETATION CONCLUSIONS: The outcome of the present investigations widened our scope on the pathogens present in ectoparasites and rodents/shrews from this area. This will help to formulate the required vector control methods to combat zoonotic diseases.

Comparative Evaluation of Different Tissues and Molecular Techniques for the Zoonotic Surveillance of Scrub Typhus.

Background and Objectives: Scrub typhus (ST) is detected in one-fourth of patients with acute febrile illnesses, confirming its nationwide re-emergence. The disease, if not diagnosed, can lead to multiple organ dysfunction and mortality. Being a vector-borne zoonotic disease, the molecular survey for pathogens in animal hosts is essential to predict the risk of its transmission to humans. Hence, this study aimed at identifying the effective animal tissue and molecular technique for zoonotic surveillance of ST infection in small animal hosts. Methods: Rodents/shrews were trapped from seventeen randomly selected villages in Puducherry between July and September, 2022. The presence of Orientia tsutsugamushi in ectoparasites and tissues including blood, lung, liver, spleen, kidney, heart, brain, and intestine retrieved from the animals was screened by nested PCR targeting 56 kDa, real-time PCR (qPCR) targeting 47 kDa and traD, and conventional PCR targeting groEL. The Weil-Felix test was carried out to detect antibodies against O. tsutsugamushi in rodent/shrew serum samples. Diagnostic accuracy measures of the molecular tests were calculated for each of the tissues by latent class modeling. Results: O. tsutsugamushi detected in the rodents/shrews were identified to be Karp-like and Kawasaki-like strains. Upon statistical analysis, qPCR targeting 47 kDa exhibited the highest accuracy measures in most of the tissues analyzed, with perfect sensitivity and specificity of 100% and 97% for intestine and lung samples for the epidemiological surveillance, respectively. Interpretation and Conclusion: The study recommends qPCR targeting 47 kDa gene and analysis of intestine and lung along with blood for the zoonotic surveillance of ST infection.

Transmission Cycle of Shimokoshi-Type Orientia tsutsugamushi in Shimane Prefecture, Japan.

To demonstrate the transmission cycle of Shimokoshi-type Orientia tsutsugamushi in Shimane Prefecture, field rodents were captured from areas where four human infections caused by the pathogen have been reported. The rodents were investigated for the transmission cycle of the pathogen based on the pathogen's genome, antibodies against the pathogen, and the vector of the pathogen (Leptotrombidium palpale). In addition, the vector was captured from the soil in the study area. A total of 44 rodents were captured. No O. tsutsugamushi DNA was detected in the blood or spleen samples by real-time polymerase chain reaction. However, a specific antibody against the pathogen was detected in 2 out of 44 (4.5%) rodents using the indirect immunoperoxidase method, indicating the presence of the pathogen in the study area. Although 29 L. palpale were identified, DNA detection was not performed because of the insufficient number of vectors, based on the DNA detection rate in previous studies. However, the identification of the vector, as well as the specific antibody in rodents, suggests the presence of the transmission cycle of Shimokoshi-type O. tsutsugamushi in Shimane Prefecture.

Clinical, laboratory profile and molecular characterization of Orientia tsutsugamushi among fatal scrub typhus patients from Karnataka, India.

BACKGROUND: Scrub typhus is a vector-borne infection caused by the obligate intracellular organism Orientia tsutsugamushi. In some cases, scrub typhus can result in severe complications, multiorgan failure and death. OBJECTIVE: To study the clinical and laboratory profiles of patients who succumbed to scrub typhus. METHODS: A prospective cohort study was conducted from August 2019 through April 2023 on scrub typhus patients admitted to our hospital. Clinical and laboratory parameters of all the patients were recorded, and blood samples were drawn. To confirm scrub typhus, a nested polymerase chain reaction (nPCR) was performed in collected samples. Viable amplicons were sequenced, and phylogenetic analyses were performed to identify infecting genotypes. RESULTS: A total of 261 patients were enrolled. Of these, nine (3.45%) patients succumbed at a median (Interquartile Range) duration of 5 (1.5, 10.5) days after admission. Sepsis with septic shock (9, 100%) and acute kidney injury (AKI) (6, 66%) were noted among the succumbed patients. All the succumbed patients (100%) required intensive care admission, inotropic and ventilatory support. While 5 (55%) patients required dialysis, two (22%) required blood transfusion. Three (33%) patient samples were co-positive for Leptospira IgM, and four (44%) patients had superinfection with Candida tropicalis, multi-drug-resistant (MDR) E. Coli sepsis, pan drug-resistant (PDR) Acinetobacter Baumanii, and Klebsiella pneumoniae. Phylogenetic analysis revealed Orientia tsutsugamushi Japanese Gilliam-variant (JG-v) like (50%), Karp-like (37.5%), and Japanese Gilliam (JG) like (12.5%) strains among succumbed patients. CONCLUSION: Delay in scrub typhus diagnosis can result in severe complications, septic shock, and multisystem organ failure, culminating in death.

Genotyping of Orientia tsutsugamushi circulating in and around Vellore (South India) using TSA 56 gene.

PURPOSE: The immunodominant TSA 56 gene of Orientia tsutsugamushi, (scrub typhus agent) has four variable regions (VD-I to VD-IV) making it useful for genotyping. This study was undertaken to determine Orientia tsutsugamushi genotypes circulating in and around Vellore using complete and partial TSA 56 gene. METHODS: Of the 162 patients positive by 47 â€‹kDa qPCR, on 21 samples PCR to amplify the complete TSA 56 gene (≈1605 bp: Long protocol) and the partial gene sequence using the Horinouchi (≈650bp) and the Furuya (≈480 bp) protocol was performed. Sanger and Nanopore sequencing was performed to obtain sequence data for assigning genotype. For 13 amplicons partial and complete gene data was obtained. RESULTS: Phylogenetic analysis of the complete gene (Long protocol) which includes VD-I to VD-IV region and partial gene (Horinouchi) which amplifies the VD-I to VD-III regions showed identical genotypes. Twelve belonged to TA763 genotype and one belongs to Karp genotype. The Furuya sequence (in silico) correctly identified the Karp genotype and 10 of the TA763 genotypes. Two TA763 genotypes (identified by complete and 650 bp partial gene analysis) were misidentified by Furuya sequence analysis as Karp genotype. CONCLUSIONS: Analysis of the 13 complete 56 â€‹kDa gene sequences suggests that TA763 is the commonest genotype in Vellore. Sanger sequencing of the 650 bp fragment gives similar results. However, these results need to be validated by larger prospective multi-centric studies.

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice.

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus.

Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.

Molecular characterization of Orientia tsutsugamushi causing fatal scrub typhus in a young male affected by so-called "mystery fever" in North India.

Scrub typhus is a zoonotic disease caused by Orientia tsutsugamushi. Although the presence of eschar is considered pathognomic, diagnosis of scrub typhus is challenging due to overlapping presentation. The diagnosis is based on the serological and molecular assay. Here, we describe a case of a young male patient who was diagnosed with scrub typhus and developed complications in the course of the disease. We also performed molecular characterization of the strain which revealed a close relatedness to Karp-like Linh.DT strains were previously reported from Vietnam.

Orientia tsutsugamushi detected by microbiome diagnostics.

Scrub typhus is caused by the mite-borne bacterium Orientia tsutsugamushi. Imported cases have been suspected in Denmark but no diagnostic method has yet been available to confirm the diagnosis. This is a case report of a 38-year-old male admitted to hospital with high fever, severe malaise and headache after returning from Malaysia. Scrub typhus was suspected and the patient recovered after one week of doxycycline treatment. The pathogen was identified by use of microbiome 16S/18S rRNA next-generation sequencing on ethylenediamine tetraacetic acid (EDTA) blood, which in the future may serve an important role in the investigation of travel-associated infections.

Orientia tsutsugamushi: A life between escapes.

The life cycle of the mite-borne, obligate intracellular pathogen Orientia tsutsugamushi (Ot), the causative agent of human scrub typhus, differs in many aspects from that of other members of the Rickettsiales order. Particularly, the nonlytic cellular exit of individual Ot bacteria at the plasma membrane closely resembles the budding of enveloped viruses but has only been rudimentarily studied at the molecular level. This brief article is focused on the current state of knowledge of escape events in the life cycle of Ot and highlights differences in strategies of other rickettsiae.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

IMPORTANCE: Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

A seminested recombinase polymerase amplification assay to detect rickettsial pathogens in clinical samples.

Treatment at the early stage of onset is vital for the prognosis of rickettsioses. But the absence of specific clinical symptoms complicates the diagnosis of this condition. Herein we established a seminested recombinase polymerase amplification assay (snRPA-nfo) that enables quick detection and differentiation of rickettsial pathogens in clinical samples with high sensitivity and specificity. The conserved 17-kDa protein gene of Rickettsia sibirica and the 47-kDa protein gene of Orientia tsutsugamushi were targeted for the duplex RPA-nfo assay. The snRPA-nfo assay exhibited an increased LOD in spiked blood samples, up to 1000-fold in comparison to standard RPA-nfo, and a better detection rate (83.3%, 5/6) than TaqMan PCR (16.6%, 1/6, Ct ≤ 35) in clinically confirmed patient blood samples. Thus, snRPA-nfo assay represents a promising alternative to TaqMan PCR in the early diagnosis of rickettsioses for point-of-care testing as well as in resource-limited settings.

The Prevalence of Rodents Orientia tsutsugamushi in China During Two Decades: A Systematic Review and Meta-Analysis.

Background: Orientia tsutsugamushi is a zoonotic intracellular pathogen that requires parasitism in eukaryotic cells to reproduce. In recent years, tsutsugamushi disease reported in many places nationwide has crossed the Yangtze River, continuously, spreading to the North China. Now this phenomenon has aroused people's attention. Materials and Methods: In this study, meta-analysis was used to analyze the infection of rodents (vectors) in China, to clarify the transmission rule of O. tsutsugamushi. Results: This study included literature from six databases (PubMed, Web of Science, Science Direct, Wanfang, CNKI, and VIP). A total of 55 articles were included in the study from 610 retrieved articles. The total infection rate of O. tsutsugamushi in rodents was 5.5% (1206/20,620, 95% confidence interval [CI]: 0.0553-0.0617). The prevalence of O. tsutsugamushi in rodents before 2013 (7.73%, 95% CI: 4.11-12.37) was higher than after 2013 (2.11%, 95% CI: 0.64-4.41). O. tsutsugamushi spread among a variety of rodents, among which Rattus losea (13.3%, 95% CI: 4.33-26.26), Rattus tanezumi (5.69%, 95% CI: 1.37-12.72), and Apodemus agrarius (5.32%, 95% CI: 2.26-9.58) infection rate was higher. Kawasaki (8.32%, 95% CI: 1.42-20.17), Karp (7.36%, 95% CI: 2.62-14.22), Kato (2.54%, 95% CI: 0.08-8.28), and Gilliam (2.13%, 95% CI: 0.42-5.09) were the main prevalent genotypes in China. The prevalence of O. tsutsugamushi in rodents was seasonal, increasing gradually in summer (2.39%, 95% CI: 0.46-5.77), peaking in autumn (4.59%, 95% CI: 1.15-10.16), and then declining. The positive rate of immunofluorescence assay (25.07%, 95% CI: 8.44-46.88) was the highest among the detection methods, and it was statistically significant (p < 0.05). Based on the subgroup of geographical factors and climatic factors, the probability of O. tsutsugamushi infection in rodents was the highest when the temperature >19℃ (8.20%, 95% CI: 1.22-20.52), the altitude <100 millimeters (7.23%, 95% CI: 3.45-12.26), the precipitation >700 millimeters (12.22%, 95% CI: 6.45-19.50), and the humidity 60-70% (7.80%, 95% CI: 4.17-12.44). Conclusions: Studies have shown that rodents carrying O. tsutsugamushi are common. People should prevent and control rodents in life and monitor rodents carrying O. tsutsugamushi for a long time.

Prevalence of chigger mites and Orientia tsutsugamushi strains in northern regions of Gangwon-do, Korea.

The present study aimed to survey the prevalence of chigger mites and Orientia tsutsugamushi (O. tsutsugamushi) infection in the northern regions of Gangwon-do, Korea. From early February to early June 2015, a total of 17,050 chiggers were collected from striped field mice, Apodemus agrarius, in Cheorwon-gun, Hwacheon-gun, Yanggu-gun, and Goseong-gun, which are well-known endemic areas of scrub typhus in Korea. The chiggers were analyzed using molecular genomic methods, as previously described. Among the 7,964 identified chiggers, the predominant species was Leptotrombidium pallidum (76.9%), followed by L. zetum (16.4%), L. orientale (4.3%), L. palpale (0.3%), L. tectum (0.2%), and Neotrombicula tamiyai (1.8%). The chigger index (CI) was highest in Hwacheon (115.58), followed by Cheorwon (97.02), Yanggu (76.88), and Goseong (54.68). Out of the 79 O. tsutsugamushi-positive chigger pools, 67 (84.8%) were identified as the Boryong strain, 10 (12.7%) as the Youngworl strain, and only 2 were the Jecheon strain. Based on the high infestation of chiggers in striped field rodents and the high rate of O. tsutsugamushi infection in chigger mites, Hwacheon-gun and Cheorwon-gun are presumed to be high-risk areas for scrub typhus. Furthermore, L. pallidum, a major vector of scrub typhus, and the dominant O. tsutsugamushi serotype, the Boryong strain, were found in the northern regions of Gangwon-do, Korea.

Brain transcriptomics reveal the activation of neuroinflammation pathways during acute Orientia tsutsugamushi infection in mice.

Scrub typhus, an acute febrile illness caused by Orientia tsutsugamushi (Ot), is prevalent in endemic areas with one million new cases annually. Clinical observations suggest central nervous system (CNS) involvement in severe scrub typhus cases. Acute encephalitis syndrome (AES) associated with Ot infection is a major public health problem; however, the underlying mechanisms of neurological disorder remain poorly understood. By using a well-established murine model of severe scrub typhus and brain RNA-seq, we studied the brain transcriptome dynamics and identified the activated neuroinflammation pathways. Our data indicated a strong enrichment of several immune signaling and inflammation-related pathways at the onset of disease and prior to host death. The strongest upregulation of expression included genes involved in interferon (IFN) responses, defense response to bacteria, immunoglobulin-mediated immunity, IL-6/JAK-STAT signaling, and TNF signaling via NF-κB. We also found a significant increase in the expression of core genes related to blood-brain barrier (BBB) disruption and dysregulation in severe Ot infection. Brain tissue immunostaining and in vitro infection of microglia revealed microglial activation and proinflammatory cytokine production, suggesting a crucial role of microglia in neuroinflammation during scrub typhus. This study provides new insights into neuroinflammation in scrub typhus, highlighting the impact of excessive IFN responses, microglial activation, and BBB dysregulation on disease pathogenesis.