Detection and Quantification of Leptospira interrogans in Hamster and Rat Kidney Samples: Immunofluorescent Imprints versus Real-time PCR

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

Ionic imbalance and lack of effect of adjuvant treatment with methylene blue in the hamster model of leptospirosis

Leptospirosis in humans usually involves hypokalaemia and hypomagnesaemia and the putative mechanism underlying such ionic imbalances may be related to nitric oxide (NO) production. We previously demonstrated the correlation between serum levels of NO and the severity of renal disease in patients with severe leptospirosis. Methylene blue inhibits soluble guanylyl cyclase (downstream of the action of any NO synthase isoforms) and was recently reported to have beneficial effects on clinical and experimental sepsis. We investigated the occurrence of serum ionic changes in experimental leptospirosis at various time points (4, 8, 16 and 28 days) in a hamster model. We also determined the effect of methylene blue treatment when administered as an adjuvant therapy, combined with late initiation of standard antibiotic (ampicillin) treatment. Hypokalaemia was not reproduced in this model: all of the groups developed increased levels of serum potassium (K). Furthermore, hypermagnesaemia, rather than magnesium (Mg) depletion, was observed in this hamster model of acute infection. These findings may be associated with an accelerated progression to acute renal failure. Adjuvant treatment with methylene blue had no effect on survival or serum Mg and K levels during acute-phase leptospirosis in hamsters

Quantification of leptospires by qPCR and the IM.

<p>A. Standard curve of the <i>lipL32</i> real-time PCR assay using DNA extracted from ten-fold serial dilutions of an <i>L. interrogans</i> strain Cop culture. Each DNA sample was quantified in duplicate and repeated twice. B. Quantification of the leptospiral load in the rat and hamster models. Rats were infected with 10⁸ leptospires and were euthanized on day 28 pi. Hamsters were inoculated with 500 leptospires (3×LD₅₀) and euthanized eight days pi. The leptospiral load in the kidneys was determined by qPCR (open symbols) and the IM (solid symbols). The leptospiral loads for the qPCR (leptospires per µg kidney DNA) and the IM (leptospires per 10 fields-of-view, ×1000 magnification) for the rat (r) and hamster (h) are presented as a scatter dot plot of the individual values for each animal, the horizontal line represents the mean value and the error bars the SEM. C. Representative examples of the imprint slides using kidney samples from an infected rat, a hamster and a non-infected control animal (magnification 1000×).</p

Comparison of culture isolation (CI), the imprint method (IM) and real-time PCR (qPCR) for the detection of leptospires in animal models simulating chronic (rat) and acute (hamster) infection.

<p>Comparison of culture isolation (CI), the imprint method (IM) and real-time PCR (qPCR) for the detection of leptospires in animal models simulating chronic (rat) and acute (hamster) infection.</p

Attenuated Nephritis in Inducible Nitric Oxide Synthase Knockout C57BL/6 Mice and Pulmonary Hemorrhage in CB17 SCID and Recombination Activating Gene 1 Knockout C57BL/6 Mice Infected with Leptospira interrogans ▿

The aims of this study were to investigate the frequency of pulmonary hemorrhage (PH) in mice unable to produce functional B and T lymphocytes and to explore the effect of an inducible nitric oxide synthase gene (Inos) knockout (KO) on the frequency/severity of interstitial nephritis in vivo. We studied the outcome of infection by the virulent Leptospira interrogans serovar Copenhageni strain Cop. The animals used were Inos KO mice, recombination activating gene 1 (Rag1) KO mice, CB17 severe combined immunodeficiency (SCID) mice, and the respective wild-type (WT) C57BL/6 and BALB/c controls. The Inos KO and WT mice survived with no clinical symptoms of leptospirosis. The frequency and severity of nephritis was significantly lower in the Inos KO mice. All of the Rag1 KO and SCID animals died of acute leptospirosis, whereas all of the WT mice survived. PH was observed in 57 and 94% of Rag1 KO mice and in 83 and 100% of SCID mice, using inoculum doses of 107 and 106 leptospires, respectively. There was no evidence of PH in the WT controls. In conclusion, the loss of the Inos gene had a negligible effect on the outcome of leptospiral infection, although we observed a reduced susceptibility for interstitial nephritis in this group. Of note, the absence of functional B- and T-cell lymphocytes did not preclude the occurrence of PH. These data provide evidence that PH in leptospirosis may not be related only to autoimmune mechanisms