Transbronchial Invasion and Proliferation of Leptospira interrogans in Lung without Inflammatory Cell Infiltration in a Hamster Model

Abstract

Leptospirosis caused by pathogenic Leptospira is one of the most common zoonoses in the world. It is believed that humans become infected with it mainly through their skin and mucous membranes by contact with water or soil that is contaminated with urine excreted from infected animals. Recently, outbreaks have frequently occurred in the tropics, especially after flooding, but how leptospires cause mass infection remains poorly understood. In this study, we injected leptospires into the tracheas of hamsters under direct view and prove for the first time that leptospires can infect through the respiratory tract. We determined that a 50% lethal dose (LD₅₀) of the Leptospira interrogans strain UP-MMC-SM (L495) for hamsters in transtracheal infection was 3.2 × 10² cells. The results of culture, macroscopic findings, and histopathological analysis suggested that intratracheally injected leptospires invaded the lung tissue, proliferated in the collagen-rich stroma adjacent to the bronchus and blood vessels, and then spread throughout the body via the bloodstream. In the lung, leptospires continuously infiltrated the alveolar wall without inflammatory cell infiltration, spread throughout the lung, and finally caused pulmonary hemorrhage. Our results revealed that the respiratory tract might be a portal of entry for leptospires. We speculate that some cases of leptospirosis might be caused by transbronchial infection from inhaling infectious aerosols containing leptospires during floods. Leptospira was also confirmed to be a unique pathogen that invades through the bronchus, proliferates in the collagen-rich lung stroma, and spreads through the alveolar interstitium throughout the lung without causing pneumonia.

Keywords: Leptospira interrogans; aerosols; bronchoalveolar lavage fluid; collagen; hamster; leptospirosis; lung; pulmonary hemorrhage; respiratory tract infection; transbronchial infection.

FIG 1

Survival (Kaplan-Meier curve) of hamsters with respiratory tract infection (A) and subcutaneous infection (B). (A) Survival of hamsters intratracheally infected with 2 × 10⁰ cells, 2 × 10¹ cells, 2 × 10² cells, 2 × 10³ cells, and 2 × 10⁴ cells of strain UP-MMC-SM (L495) (5 hamsters in each dose group); (B) survival of hamsters subcutaneously infected with 1 × 10⁰ cells, 1 × 10¹ cells, 1 × 10² cells, 1 × 10³ cells, and 1 × 10⁴ cells of strain L495 (5 hamsters in each dose group).

FIG 2

Changes in the number of leptospires in each organ and specimen after respiratory tract infection by the limiting dilution culture method on 96-well plates. A 200-μl leptospire suspension containing 2 × 10⁴ cells of strain UP-MMC-SM (L495) was instilled into the tracheas of 7-week-old male hamsters. Infected hamsters were euthanized on days 0 (i.e., 2 h) to 9 postinfection every day (3 per day). The number of leptospires in each homogenized organ (right lung [A], a subsegment of the liver [segment 4] [B], and right kidney [C]) and specimen (blood [D], urine [E], and BALF [F]) was calculated by the limiting dilution culture method on 96-well plates. The dots indicate the number of leptospires in each individual hamster. The dashed lines indicate the detection limit of leptospires.

FIG 3

Macroscopic lesions in hamsters intratracheally infected with strain UP-MMC-SM (L495). Pulmonary hemorrhage (A), renal hemorrhage (B), and intestinal hemorrhage and jaundice (C).

FIG 4

Changes in the numbers and fractions of leucocytes in the BALF of intratracheally infected hamsters. A 200-μl leptospire suspension containing 2 × 10⁴ cells of strain UP-MMC-SM (L495) was instilled into the tracheas of 7-week-old male hamsters. Infected hamsters were euthanized on days 0 (i.e., 2 h) to 9 postinfection every day (3 per day). The number of leukocytes in the BALF was counted using a hemacytometer, and the leukocyte fractions in the BALF were determined by Wright-Giemsa staining. As a noninfected control, BALF was collected from hamsters intratracheally administered 200 μl of only PBS on day 0 (n = 2).

FIG 5

Leptospiral infiltration in the lung after respiratory tract infection and subcutaneous infection. (A) Hamsters intratracheally infected with 2 × 10⁶ cells of strain UP-MMC-SM (L495) were euthanized at 0, 2, 4, 6, 7, 8, and 9 days after infection (2 per day), and the left upper lungs were collected, fixed, cut into 4-μm slices (three serial sections), and then stained with HE, immunofluorescence stain, and EVG stain. Representative microscope images of the serial sections at 0, 2, 4, 6, and 7 days postinfection are shown. Hamsters subcutaneously infected with 1 × 10⁶ cells of strain L495 were euthanized at 0, 2, 4, 6, 7, 8, and 9 days postinfection (2 per day), and the left upper lungs were collected, fixed, cut into 4-μm slices (three serial sections), and then stained with HE, immunofluorescence stain, and EVG stain. Representative microscope images of the serial sections at only 7 and 8 days postinfection are shown because no changes were observed on days 0 to 6 postinfection. For immunofluorescence staining, leptospires were stained with rabbit anti-Leptospira interrogans serovar Manilae antiserum and goat anti-rabbit IgG antibody labeled with Alexa Fluor 488 (green), and the nuclei of lung cells were stained with DAPI (blue). In EVG staining, elastic fibers and cell nuclei were stained black, collagen fibers were stained red, and other tissues were stained yellow. The areas boxed by black frames in the HE-stained images are enlarged in panel B. The area boxed by a white frame in the immunofluorescence stain of day 2 in panel B is enlarged in Fig. 6. Scale bars, 200 μm (A) and 20 μm (B).

FIG 6

Enlarged immunofluorescence stain image of lung tissue after respiratory tract infection. The area boxed by a white frame in the immunofluorescence stain of day 2 in Fig. 5B is enlarged. Leptospires were stained with rabbit anti-Leptospira interrogans serovar Manilae antiserum and goat anti-rabbit IgG antibody labeled with Alexa Fluor 488 (green), and the nuclei of lung cells were stained with DAPI (blue). The spiral structure characteristic of leptospires was confirmed. Immunofluorescence staining in the lung on day 2 postinfection was observed with a confocal laser scanning microscope. Scale bar, 20 μm.

FIG 7

Transmission electron microscope (TEM) images of the lung at the end of intratracheal infection with leptospires. (A) The lower left lung at 7 days postinfection was analyzed by TEM. Leptospires (white arrow) were phagocytosed by alveolar macrophages, covered with a membrane, and swirling. (B) There were leptospires near the collagen fibers (white stars) in the intercellular spaces of the peripheral alveolar stroma. (B, C) Shadowing was observed around the leptospires, suggesting that the leptospire surface may have been coated with a substance. Scale bars, 5 μm (A), 1 μm (B), and 2 μm (C).

FIG 8

Scanning electron microscope-correlative light and electron microscope (SEM-CLEM) images of the lung at the end of intratracheal infection with leptospires. The lower right lung of an infected hamster at 7 days postinfection was cut by the cross-cutting method, and the surface was observed by SEM-CLEM. Leptospires were stained with rabbit anti-Leptospira interrogans serovar Manilae antiserum and goat anti-rabbit IgG antibody labeled with Alexa Fluor 488 (green) (images on the bottom left of each panel). The data taken with an SEM (images on the top left of each panel) after fluorescence staining were edited (images on the right of each panel) using Photoshop CC (overall view [A], enlarged view of the area surrounded by a white box in panel A [B]). Leptospira cells were observed on the surfaces of separated alveolar cells exposed by dissociation of intercellular adhesion with the cross-cutting method. Scale bars, 10 μm (A) and 1 μm (B).

FIG 9

Summary of the processes of leptospiral infiltration of the lungs of hamsters with respiratory tract infection and subcutaneous infection.