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, 8 (11), e3307
eCollection

Kinetics of Leptospira Interrogans Infection in Hamsters After Intradermal and Subcutaneous Challenge

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Kinetics of Leptospira Interrogans Infection in Hamsters After Intradermal and Subcutaneous Challenge

Mariana L Coutinho et al. PLoS Negl Trop Dis.

Abstract

Background: Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection.

Methodology/principal findings: Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7.

Conclusions/significance: The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2×106 leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis.

Conflict of interest statement

We have read the journal's policy and the authors of this manuscript have the following competing interests: DAH and JM have intellectual property involving leptospiral proteins, none of which are described in this manuscript. This does not alter our adherence to all PLOS policies on sharing data and materials.

Figures

Figure 1
Figure 1. Kinetics of weight and kidney histopathology and function during leptospirosis infection.
Animals were weighed at the time of subcutaneous (panels A and C) and intradermal (panels B and D) challenge (day zero) and daily thereafter. Panels A and B show mean change in weight relative to the previous day compared with the mean total serum protein (TSP) concentration showing weight loss and hemoconcentration beginning on day 6. Panels C and D show mean renal histopathology scores and mean serum creatinine, showing that abnormal histopathology preceded changes in kidney function tests. Error bars show standard deviation of the mean. Vertical dashed line indicates the point at which animals began to meet endpoint criteria. The number of asterisks indicate the level of significance compared to the control group (*P≤0.05, ** P≤0.01, ***P≤0.001, ****P≤0.0001). There were no differences between hamsters challenged subcutaneously and intradermally except for % weight change on day 7 after challenge.
Figure 2
Figure 2. Kinetics of inflammatory markers and liver enzymes.
Blood samples were collected at the time of subcutaneous (panels A and C) and intradermal (panels B and D) challenge (day zero) and daily thereafter. Panels A and B show mean absolute neutrophil counts (ANC) and vascular endothelial growth factor (VEGF) showing that the elevation in ANC on day 5 preceded the elevation in VEGF on day 6. Panels C and D show that the serum alkaline phosphatase and glutamic pyruvate transaminase (SGPT) peaked on day 6. Error bars show standard deviation of the mean. Vertical dashed line indicates the point at which animals began to meet endpoint criteria. The number of asterisks indicate the level of significance compared to the control group (*P≤0.05, ** P≤0.01, ***P≤0.001, ****P≤0.0001). There were no differences between hamsters challenged subcutaneously and intradermally except for the ANC on day 7 after challenge.
Figure 3
Figure 3. Leptospiral tissue burden.
Kidney (panel A) and liver (panel B) tissues were subjected to DNA extraction and real-time PCR to measure the leptospiral copies per microgram of tissue DNA. Means (bars) and actual values (circles or diamonds) are depicted for animals challenged subcutaneously or intradermally. Vertical dashed line indicates the point at which animals began to meet endpoint criteria. The number of asterisks indicate the level of significance compared to the control group (*P≤0.05, ** P≤0.01, ***P≤0.001, ****P≤0.0001). Leptospiral tissue burdens after subcutaneous challenge were significantly higher than after intradermal challenge on day 6 in both the kidney and liver (P<0.01).
Figure 4
Figure 4. Histopathology changes in the kidney and perinephric tissues.
A. Inflammatory changes in perinephric fat. B. Inflammatory changes in periureteral fat. C. Infiltration of inflammatory cells beneath the epithelium of the renal pelvis. D. Massively enlarged perihilar lymph node. E. Early glomerular and tubular damage with intratubular hyaline cast formation. F. Early interstitial inflammation in the kidney. G. Diffuse glomerular and tubular damage with hyaline cast formation. H. Severe tubular damage.
Figure 5
Figure 5. Antibody response in infected hamsters.
Hamster immunoglobulin responses to heat-killed leptospires were measured by ELISA. Mean and standard errors are shown for serum samples (tested in triplicate at a dilution of 1∶6400) obtained from hamsters challenged intradermally (red) or subcutaneously (blue). Dotted gray line represents two standard deviations above background ELISA for sera from six uninfected control hamsters. Vertical dashed line indicates the point at which animals began to meet endpoint criteria. The number of asterisks indicate the level of significance compared to the control group (*P≤0.05, ***P≤0.001). Leptospiral antibody levels after intradermal challenge were significantly higher than after subcutaneous challenge on day 8 (P<0.05).
Figure 6
Figure 6. Fold change in kidney burden.
The fold change in kidney burden relative to the previous day as determined by qPCR was plotted for animals challenged intradermally (ID) vs. subcutaneously (SQ). There was a one-day surge in the burden of organisms in the kidney several days after challenge that was larger and later in animals challenged via the SQ route as compared to the ID route.

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