Pathological Responses in Asian House Shrews (Suncus murinus) to the Naturally Acquired Orientia tsutsugamushi Infection

Abstract

Scrub typhus is a re-emerging disease caused by Orientia tsutsugamushi, transmitted by mites belonging to the family Trombiculidae. Humans and rodents acquire the infection by the bite of larval mites/chiggers. Suncus murinus, the Asian house shrew, has been reported to harbor the vector mites and has been naturally infected with O. tsutsugamushi. The present study aimed to localize and record O. tsutsugamushi in the tissues and the host response in shrews naturally infected with O. tsutsugamushi. Sheehan's modified May-Grunwald Giemsa staining was carried out in 365 tissues from 87 animals, and rickettsiae were documented in 87 tissues from 20 animals. Immunohistochemical (IHC) staining, using polyclonal antibodies raised against selected epitopes of the 56-kDa antigen, was carried out, and 81/87 tissue sections were tested positive for O. tsutsugamushi. By IHC, in addition to the endothelium, the pathogen was also demonstrated by IHC in cardiomyocytes, the bronchiolar epithelium, stroma of the lungs, hepatocytes, the bile duct epithelium, the epithelium and goblet cells of intestine, the tubular epithelium of the kidney, and splenic macrophages. Furthermore, the pathogen was confirmed by real-time PCR using blood (n = 20) and tissues (n = 81) of the IHC-positive animals. None of the blood samples and only 22 out of 81 IHC-positive tissues were tested positive by PCR. By nucleotide sequencing of the 56-kDa gene, Gilliam and Karp strains were found circulating among these animals. Although these bacterial strains are highly virulent and cause a wide range of pathological alterations, hence exploring their adaptive mechanisms of survival in shrews will be of significance. Given that the pathogen localizes in various organs following a transient bacteremia, we recommend the inclusion of tissues from the heart, lung, intestine, and kidney of reservoir animals, in addition to blood samples, for future molecular surveillance of scrub typhus.

Keywords: Orientia tsutsugamushi; Suncus murinus; histochemistry; immunohistochemistry; pathological responses; scrub typhus.

Figure 1

Schematic representation of H&E ×400, Giemsa–Sheehan’s modified May–Grunwald ×400 and IHC immunoperoxidase/DAB substrate/Harris hematoxylin ×400 staining techniques carried out on the heart tissue of S. murinus. The purple- and brown-colored cocco-bacillary structures in the insets indicate the Rickettsia and Orientia tsutsugamushi localized in the tissue by the Giemsa–Sheehan’s modified May–Grunwald and immunohistochemical staining techniques respectively.

Figure 2

Representative heart tissue samples of shrews stained positive for Rickettsia by Giemsa–Sheehan’s modified May–Grunwald stain appearing as reddish-purple-color in the endothelium of blood vessels and in the cytoplasm of cardiomyocytes ×400 (a); endothelial cells of pulmonary blood vessels ×400 (b); in peribronchiolar infiltrates in lung ×400 (c); endothelial cells of central vein of liver ×400 (d); in stromal cells of portal triad ×400 (e); Glisson’s capsule ×400 (f); in splenic macrophages ×400 (g); intestinal epithelial cells and goblet cells ×400 (h); in renal tubular epithelium ×400 (i) and endothelial cells of renal blood vessels ×400 (j). Insets shows digitally enhanced views of the original image (×400) to indicate the rickettsial organims.

Figure 2

Representative heart tissue samples of shrews stained positive for Rickettsia by Giemsa–Sheehan’s modified May–Grunwald stain appearing as reddish-purple-color in the endothelium of blood vessels and in the cytoplasm of cardiomyocytes ×400 (a); endothelial cells of pulmonary blood vessels ×400 (b); in peribronchiolar infiltrates in lung ×400 (c); endothelial cells of central vein of liver ×400 (d); in stromal cells of portal triad ×400 (e); Glisson’s capsule ×400 (f); in splenic macrophages ×400 (g); intestinal epithelial cells and goblet cells ×400 (h); in renal tubular epithelium ×400 (i) and endothelial cells of renal blood vessels ×400 (j). Insets shows digitally enhanced views of the original image (×400) to indicate the rickettsial organims.

Figure 3

Representative images of the IHC positive immunostaining (IHC immunoperoxidase/DAB substrate/Harris hematoxylin) of Orientia antigen, visualized as brown-color in the endothelium of endocardium ×400 (a); in cardiomyocytes (inset) ×400 (b); endothelium of pulmonary blood vessel (open arrow) and in peribronchiolar infiltrates ×400 (arrow) (c) and within alveolar macrophages ×1000 (inset) (d); in liver—endothelium of central vein ×200 (inset) (e), in bile duct epithelium ×200 (inset) (f) and within hepatocytes ×400 (inset) (g); in splenic sinusoids ×200 (inset) (h) and in splenic macrophages ×200 (inset) (i); in intestinal epithelial cell ×400 (inset) (j,k); in renal tubular epithelium (arrow) and in endothelium of renal blood vessels (open arrow) ×200 (inset) (l).

Figure 3

Representative images of the IHC positive immunostaining (IHC immunoperoxidase/DAB substrate/Harris hematoxylin) of Orientia antigen, visualized as brown-color in the endothelium of endocardium ×400 (a); in cardiomyocytes (inset) ×400 (b); endothelium of pulmonary blood vessel (open arrow) and in peribronchiolar infiltrates ×400 (arrow) (c) and within alveolar macrophages ×1000 (inset) (d); in liver—endothelium of central vein ×200 (inset) (e), in bile duct epithelium ×200 (inset) (f) and within hepatocytes ×400 (inset) (g); in splenic sinusoids ×200 (inset) (h) and in splenic macrophages ×200 (inset) (i); in intestinal epithelial cell ×400 (inset) (j,k); in renal tubular epithelium (arrow) and in endothelium of renal blood vessels (open arrow) ×200 (inset) (l).

Figure 4

Comparison of histochemical and IHC staining for localization of Rickettsia/Orientia in the tissues of Suncus murinus.

Figure 5

Phylogenetic clustering of the isolates of Orientia tsutsugamushi based on the 56-kDa nucleotide sequences. The phylogenetic tree was constructed using the maximum likelihood method with 1000 bootstrap replicates on Mega X platform. The solid triangle symbol indicates the isolates from the current study.