Adipose tissue is the first colonization site of <i>Leptospira interrogans</i> in subcutaneously infected hamsters

<div><p>Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an <i>in vivo</i> imaging system to observe the whole bodies of animals infected with <i>Leptospira interrogans</i> and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 10⁴ bioluminescent leptospires, were analyzed by <i>in vivo</i> imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.</p></div

Bioluminescence dissemination of <i>Leptospira</i> in hamsters.

<p>(A) The survival rate of Golden Syrian hamsters (n = 8) infected subcutaneously with 10⁴ <i>L</i>. <i>interrogans</i> strain M1307 into the right inguinal region, and representative ventral view photographic images tracking the hamster infections on different days post-infection. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm²/sr as indicated on the scales, where red is the most intense (3×10⁵) and purple is the least intense (3×10⁴). (B,C) Average luminescence intensities in each ROI of injection site (B) and abdominal center (C) at different days post-infection. Data are expressed as the means ± SEM of total flux in photons/second in each ROI in eight infected hamsters (●) and two uninfected controls (◦). <i>p</i> values (*<i>p</i><0.05), between groups.</p

Bioluminescence changes in hamster organs.

<p>Representative bioluminescence images (ventral view) from M1307-infected hamsters at each phase. Images represent subcutaneous tissues after skin incision and organs after laparotomy, as well as <i>ex vivo</i> organs (blood plus liver and kidney cross sections). The scale is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172973#pone.0172973.g001" target="_blank">Fig 1</a>.</p

<i>Leptospira</i> distribution in skin and subcutaneous tissue.

<p>Representative light field (A, C) and fluorescence images (B, D) of the skin and subcutaneous tissue (A, B) or adipose tissue (C, D) around the injection sites of M1307 collected from infected hamsters at phase 4. Fluorescence images (B, D) showing cell nuclei stained with DAPI (blue), autofluorescence of the skin and subcutaneous tissue (green, not shown in panel D), and leptospires stained with rabbit polyclonal antiserum and Cy5-conjugated anti-rabbit monoclonal antibody (red). The framed area in (B) is enlarged at the upper right. Scale bars: 100 μm (A, B), 500 μm (C, D).</p

Transmission electron microscopy of adipose tissue blood vessels.

<p>Representative transmission electron microscope images of subcutaneous adipose tissue blood vessels around the injection sites of <i>Leptospira</i>-infected hamsters at phase 4. The framed area in (A) is enlarged in (B). The scale bars represent 5 μm (A) and 1 μm (B). The arrowheads point to <i>Leptospira</i> and the arrows show the red blood cells.</p