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 10 7 and 10 6 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.
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.
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