Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis

Institut Pasteur, Unité de Biologie des Spirochètes, Paris, France.
Journal of Medical Microbiology (Impact Factor: 2.25). 06/2009; 58(Pt 5):648-55. DOI: 10.1099/jmm.0.008169-0
Source: PubMed


The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD(50), rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5x10(4) leptospires ml(-1). The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

Full-text preview

Available from:
  • Source
    • "Of the 13 species investigated , the seroprevalence rate and the prevalence of renal infection were highest in black rats (78.8% and 65.7%, respectively) while the renal leptospiral load in this species showed unexpectedly high variations. The dynamics of leptospirosis infection have not yet been well studied: some studies attempted to reproduce renal colonization under experimental conditions, in particular in the rat or in guinea pig models (Athanazio et al., 2008; Bonilla-Santiago and Nally, 2011; Lourdault et al., 2009; Thiermann, 1981), whereas other authors focused on reproducing the acute disease in sensitive species such as mice (Santos et al., 2010) or primates (Pereira et al., 2005). Nevertheless, it is acknowledged that experimental studies cannot replicate field conditions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In 2009, a survey was conducted in Reunion Island to determine the renal leptospiral load in black rats trapped in the field. The concentration of leptospires in kidney tissue was calculated using qPCR. Our results showed high inter-individual variations of renal bacterial load in naturally infected black rats (mean=8.27±4.72 log-genome copies per mg kidney tissue). The objective of this study was to model the renal leptospiral load in 50 naturally infected black rats as a function of sex, age, and weight. Statistical analysis by sex showed that, in naturally infected males, the renal leptospiral load was correlated with weight (p-value=0.032). Moreover, our model showed that weight and sex were significant explanatory variables for the renal leptospiral load in naturally infected young black rats (R(2)=0.953). Laboratory experimentation could not replicate naturally acquired infection, but field studies also present many limitations. Our study is the first attempt to explain individual variations in the renal leptospiral load in naturally infected reservoir animals but further research is needed.
    Acta tropica 12/2012; 125(3). DOI:10.1016/j.actatropica.2012.11.011 · 2.27 Impact Factor
  • Source
    • "The lipL32 gene, which encodes the immunodominant lipoprotein located in the leptospiral outer membrane, is highly conserved among the pathogenic serovars and is absent in the saprophytes [16], [17]. These assays have been used to monitor renal colonization in experimental infection [15], [18], to evaluate urinary shedding of leptospires in dogs [19] and for case confirmation in human subjects during outbreak investigations [20], [21], [22]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.
    PLoS ONE 02/2012; 7(2):e32712. DOI:10.1371/journal.pone.0032712 · 3.23 Impact Factor
  • Source
    • "The results indicated that leptospirosis is rarely transmitted by the bite of rat [11]. Since then, different infection routes such as conjunctival (c.j.) and subcutaneous (s.c.) have been employed in canine, horse, hamster and guinea pig, and resulting in acute leptospirosis in inoculated animals [11-16]. By using infection routes different from the classic i.p. inoculation, these studies contributed to the elucidation of pathogenesis of leptospirosis in experimental animals. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leptospires are presumed to enter their host via small abrasions or breaches of the skin. The intraperitoneal route, although commonly used in guinea pig and hamster models of leptospirosis, does not reflect conditions encountered during natural infection. The aim of this study is to develop a novel leptospirosis guinea pig model through epicutaneous route and to elucidate the pathogenesis of leptospirosis in experimental guinea pigs by comparing the data from other studies using different infection routes. The guinea pigs were inoculated with 5 × 108 Leptospira interrogans strain Lai onto either shaved-only or abraded skin. The guinea pigs were sacrificed at 2, 8, 24, 48, 72, 96 and 144 h post-infection (p.i.) followed by harvest of the lungs, liver, kidneys, spleen, and the skin around the inoculated sites for further examinations. Hematoxylin and eosin (HE) staining and electron microscopy were used to detect the pathologic changes. Real time PCR and immunohistochemistry staining were performed to detect dynamic distribution of leptospires in blood and tissues, respectively. In the guinea pigs with abraded skin inoculations, leptospires were detected in blood as early as 2 h post infection (p.i.) and then disseminated to the liver, lungs and kidneys of almost all animals within 96 h p.i.. Leptospires were also detected engulfed in the swelling vascular endothelial cells and were frequently aggregated around the capillaries in the dermis and subcutaneous tissue under the inoculated site. For the guinea pigs with abraded skin inoculations, hemorrhage at the dermis around the inoculated site was found before the appearance of internal organs hemorrhage, severe lesions such as hemorrhages in the lungs, nephritis, jaundice, haematuria were also observed, and two of seven guinea pigs died at 144 h p.i. while no lesions and leptospires were detected in the shaved-only guinea pigs using the same dose of strain Lai. Intact keratinocyte layer is a very efficient barrier against leptospires, and intact skin can prevent the infiltration of leptosipres to the host. Leptospires can penetrate abraded skin and quickly establish a systemic infection by crossing tissue barriers. We have successfully established a novel leptospirosis guinea pig model through epicutaneous inoculations route, which replicates a natural course of infection and appears to be an alternative way to investigate the pathogenesis of leptospirosis, especially in terms of early stage of host-pathogen interactions. This novel model may also be advantageous for studies of the mechanisms involved in cutaneous barriers and epidermal interactions with this organism.
    BMC Infectious Diseases 01/2012; 12(1):20. DOI:10.1186/1471-2334-12-20 · 2.61 Impact Factor
Show more