An Intradermal Inoculation Model of Scrub Typhus in Swiss CD-1 Mice Demonstrates More Rapid Dissemination of Virulent Strains of Orientia tsutsugamushi

Piyanate Sunyakumthorn,Alison Luce-Fedrow,Teik-Chye Chan,Margaret Jones,Gareth D H Turner,Tippawan Anantatat,Daniel H Paris,Nicholas P J Day,Ju Jiang,Suchismita Chattopadhyay,Allen L Richards,Roman Ganta

doi:10.1371/journal.pone.0054570

Abstract

Scrub typhus is an important endemic disease of the Asia-Pacific region caused by Orientia tsutsugamushi. To develop an effective vaccine to prevent scrub typhus infection, a better understanding of the initial host-pathogen interaction is needed. The objective of this study was to investigate early bacterial dissemination in a CD-1 Swiss outbred mouse model after intradermal injection of O. tsutsugamushi. Three human pathogenic strains of O. tsutsugamushi (Karp, Gilliam, and Woods) were chosen to investigate the early infection characteristics associated with bacterial virulence. Tissue biopsies of the intradermal injection site and draining lymph nodes were examined using histology and immunohistochemistry to characterize bacterial dissemination, and correlated with quantitative real-time PCR for O. tsutsugamushi in blood and tissue from major organs. Soluble adhesion molecules were measured to examine cellular activation in response to infection. No eschar formation was seen at the inoculation site and no clinical disease developed within the 7 day period of observation. However, O. tsutsugamushi was localized at the injection site and in the draining lymph nodes by day 7 post inoculation. Evidence of leukocyte and endothelial activation was present by day 7 with significantly raised levels of sL-selectin, sICAM-1 and sVCAM-1. Infection with the Karp strain was associated with earlier and higher bacterial loads and more extensive dissemination in various tissues than the less pathogenic Gilliam and Woods strains. The bacterial loads of O. tsutsugamushi were highest in the lungs and spleens of mice inoculated with Karp and Gilliam, but not Woods strains. Strains of higher virulence resulted in more rapid systemic infection and dissemination in this model. The CD-1 mouse intradermal inoculation model demonstrates features relevant to early scrub typhus infection in humans, including the development of regional lymphadenopathy, leukocyte activation and distant organ dissemination after low-dose intradermal injection with O. tsutsugamushi.

Highlights

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi, a Gram-negative intracellular bacterium transmitted by larval trombiculid mites known as chiggers, which serve as both disease vectors and reservoirs for maintenance of O. tsutsugamushi in nature [1]
Scrub typhus is endemic in the Asian-Pacific region, where up to 28% of diagnosed febrile illnesses among hospitalized patients are due to scrub typhus, and case fatality rates can rise to 50% in untreated patients [2,3,4,5,6]
The control mouse sera indicated that antibodies against O. tsutsugamushi were detected in the positive controls; O. tsutsugamushi ID inoculated mice evaluated in this study did not mount a detectable antibody titer within the 7 day observation period

Summary

Introduction

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi, a Gram-negative intracellular bacterium transmitted by larval trombiculid mites known as chiggers, which serve as both disease vectors and reservoirs for maintenance of O. tsutsugamushi in nature [1]. Various murine models of scrub typhus infection have been developed, and the CD-1 outbred Swiss mouse model is widely used to study host immune response and vaccine development [7,8]. Outbred mice demonstrate broader and more heterogeneous immune responses that more accurately reflect the natural and vaccine induced immune responses as well as the associated immunopathophysiology in the human host [8,9]. Intraperitoneal (IP) and intravenous (IV) injections are commonly used as routes of infection for O. tsutsugamushi in laboratory animals. They are not the natural route by which vertebrate hosts acquire O. tsutsugamushi infection in nature. In cynomolgus primates and humans, early dissemination of O. tsutsugamushi via hematogeneous and/or lymphatic system was accompanied by regional lymphadenopathy and subsequent systemic dissemination and onset of clinical manifestations [13,14,15]

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Conclusion