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.

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    • "En los últimos años, el protocolo de Merien et al (1992) fue evaluado mediante una técnica de PCR en tiempo real utilizando química SYBR Green. En el campo de investigación, Lourdault et al (2009) evaluaron la carga bacteriana en sangre y órganos (riñón, hígado, bazo, etc.) empleando un modelo de infección en cobayos. Mientras que, Fornazari et al (2012) compararon una PCR punto final, con una PCR cuantitativa en tiempo real, cultivo bacteriológico y la técnica de Warthin Starry para el diagnóstico de Leptospira spp en muestras de sangre y órganos (riñón e hígado) de ovinos y caprinos serológicamente reactivos; demostrando que la PCR cuantitativa en tiempo real fue el método más sensible para el diagnóstico. "

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    • "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. "
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    • "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]. "
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