Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

Abstract

Background: Severe leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.

Methodology/principal findings: Male Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6-7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.

Conclusion/significance: This study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.

Fig 1. Gross appearance of organs.

Petechial haemorrhages were observed in the lungs from day 1 p.i. and became severe from day 3 p.i. onwards. Kidneys became progressively pale from day 6 p.i. onwards. No notable changes were observed in the liver. C: Control; D1-D7: Day 1 to Day 7. The arrow shows a petechial haemorrhage.

Fig 2. Gross finding in dissected areas.

The yellowish discolouration was observed on adipose tissue on day 6 p.i. in infected hamsters. C: control; D6: day 6 p.i.

Fig 3. Histopathological lesions in the lung of infected hamsters.

Congestion and haemorrhage occurred as early as day 1 p.i. while dilated and collapsed alveoli occurred on days 6 and 7 p.i. and in dead hamsters. CG: Congestion, H: Haemorrhage, CA: Collapsed alveoli, ST: Septal thickening, DA: Dilated alveoli. C: Control, D1-D7: Day 1- day 7 and DD: Dead. Magnification: x100, bar: 100 μm.

Fig 4. Histopathological lesions in the liver of infected hamsters.

Congestion occurred as early as day 1 p.i. while infiltration of inflammatory cells, disorganized hepatocyte cords and swelling of hepatocytes occurred on day 4 p.i. onwards. SC: Sinusoid congestion, IC: Infiltration of inflammatory cells, DHC: Disorganized hepatocyte cords, SH: Swollen hepatocytes and H: Haemorrhage. C: Control, D1-D7: Day 1- day 7 and DD: Dead. Magnification: x100, bar: 100 μm.

Fig 5. Histopathological lesions in kidneys of infected hamsters.

Congestion occurred as early as day 3 p.i., haemorrhage, infiltration of inflammatory cells, dilation and collapse of Bowman’s space on day 4 p.i. and dilation of tubule and degeneration of epithelial cells in proximal and distal tubules occurred on day 5 p.i. onwards. CG: Congested glomerulus, CB: Collapse of Bowman’s space, DB: Dilated Bowman’s space, DT: Dilated tubule, DPD: Degeneration of epithelial cells in proximal and distal tubules, IC: Infiltration of inflammatory cells, and H: Haemorrhage. C: Control, D1-D7: Day 1- day 7 and DD: Dead. Magnification: x100, bar: 100 μm.

Fig 6. Leptospiral load in blood and organs tissues of hamsters infected with L. interrogans HP358.

D1-D7: day 1 to day 7; DD: dead hamsters. The leptospiral copies number in blood and organs can be found in S1 Table.

Fig 7. The pattern of WBC, neutrophils, monocytes, lymphocytes and platelets levels in control (blue line) and infected hamsters (red line).

D1-D7: Day 1 to day 7. *P≤0.05, **P≤0.01, ***P≤0.001.

Fig 8. Serum level of total bilirubin, direct bilirubin, CK, ALT, AST, creatinine and urea in control (blue line) and infected hamsters (red lines).

The biochemical measurements convey the function state of the liver and kidney. D1-D7: Day 1 to Day 7. *P≤0.05, **P≤0.01, ***P≤0.001.

Fig 9. Modulation of pro-inflammatory cytokines and enzyme in the blood of control (blue line) and infected hamsters (red line).

Total RNA was extracted from whole blood on day 1 and days 3 to 7 p.i.. D1-D7: Day 1 to day 7. *P≤0.05. The fold gene expression value can be found in S2 Table.

Fig 10. Modulation of pro-inflammatory cytokines and enzymes in the lungs of control (blue line) and infected hamsters (red line).

Total RNA was extracted from lungs on day 1 p.i. and days 3 to 7 p.i. D1-D7: Day 1 to day 7. DD: dead. **P≤0.01, ***P≤0.001. The fold gene expression value can be found in S2 Table.

Fig 11. Modulation of pro-inflammatory cytokines and enzymes in the liver of control (blue line) and infected hamsters (red line).

Total RNA was extracted from liver on day 1 p.i. and days 3 to 7 p.i. D1-D7: Day 1 to day 7. DD: dead. *P≤0.05, **P≤0.01, ***P≤0.001.The fold gene expression value can be found in S2 Table.

Fig 12. Modulation of pro-inflammatory cytokines and enzymes in the kidneys of control (blue line) and infected hamsters (red line).

Total RNA was extracted from kidneys on day 1 p.i. and days 3 to 7 p.i. D1-D7: Day 1 to day 7. DD: dead. *P≤0.05, **P≤0.01, ***P≤0.001. The fold gene expression value can be found in S2 Table.

Fig 13. Modulation of chemokines in blood, lungs, liver and kidneys of control (blue line) and infected hamsters (red line).

D1-D7: Day 1 to day 7. DD: dead *P≤0.05, **P≤0.01, ***P≤0.001. Control. The fold gene expression value can be found in S3 Table.

Fig 14. Expression of TGF-β1 in blood, lungs, liver and kidneys of control (blue line) and infected hamsters (red line).

D1-D7:Day 1 to day 7. DD: dead. *P≤0.05, **P≤0.01, ***P≤0.001.The fold gene expression value can be found in S4 Table.