Flávia W. Cruz McBride’s research while affiliated with Catholic University of Pelotas and other places

Determination of the ED50 in Syrian hamsters. Groups of hamsters (n = 3) were inoculated with a range of infective doses of L. interrogans serovar Copenhageni strain Fiocruz L1-130, see S1 Table. Three independent experiments were carried to calculate the ED50. The number of leptospires/infective dose and hamster survival are shown. (TIF)

Conservation of LigB and LigB(131–645) among the pathogenic Leptospira spp. The paired columns represent the mean amino acid pairwise identity of LigB and LigB(131–645) from L. interrogans serovar Copenhageni strain L1-130, respectively, with the corresponding proteins from pathogenic Leptospira spp. The error bars represent the standard deviation from the mean. (TIF)

Determination of the ED50 for L. interrogans strain Fiocruz L1-130 in the Syrian hamster model of leptospirosis. (DOCX)

Neglected tropical diseases, including zoonoses such as leptospirosis, have a major impact on rural and poor urban communities, particularly in developing countries. This has led to major investment in antipoverty vaccines that focus on diseases that influence public health and thereby productivity. While the true, global, impact of leptospirosis is unknown due to the lack of adequate laboratory diagnosis, the WHO estimates that incidence has doubled over the last 15 years to over 1 million cases that require hospitalization every year. Leptospirosis is caused by pathogenic Leptospira spp. and is spread through direct contact with infected animals, their urine or contaminated water and soil. Inactivated leptospirosis vaccines, or bacterins, are approved in only a handful of countries due to the lack of heterologous protection (there are > 250 pathogenic Leptospira serovars) and the serious side-effects associated with vaccination. Currently, research has focused on recombinant vaccines, a possible solution to these problems. However, due to a lack of standardised animal models, rigorous statistical analysis and poor reproducibility, this approach has met with limited success. We evaluated a subunit vaccine preparation, based on a conserved region of the leptospiral immunoglobulin-like B protein (LigB(131–645)) and aluminium hydroxide (AH), in the hamster model of leptospirosis. The vaccine conferred significant protection (80.0–100%, P < 0.05) against mortality in vaccinated animals in seven independent experiments. The efficacy of the LigB(131–645)/AH vaccine ranged from 87.5–100% and we observed sterile immunity (87.5–100%) among the vaccinated survivors. Significant levels of IgM and IgG were induced among vaccinated animals, although they did not correlate with immunity. A mixed IgG1/IgG2 subclass profile was associated with the subunit vaccine, compared to the predominant IgG2 profile seen in bacterin vaccinated hamsters. These findings suggest that LigB(131–645) is a vaccine candidate against leptospirosis with potential ramifications to public and veterinary health.

Endpoint IgG GMTs of the LigB(131–645) and bacterin vaccine preparations. The endpoint titres were determined for sera collected from hamsters 14 days PV with LigB(131–645), and the bacterin vaccine in an ELISA based on rLigB(131–645). A-C) serial dilution of PI and PV sera collected from hamsters immunised with rLigB(131–645) in three independent experiments. D) a representative experiment for hamsters immunized with a bacterin vaccine is shown. The standard deviation (vertical bars) for each data point is shown. (TIF)

Protection conferred by immunization with rLigB(625–1259) and rLigB(131–645) against lethal challenge in the hamster model of leptospirosis. (DOCX)

Conservation of LigB and LigB(131–645) among the pathogenic Leptospira spp. (DOCX)

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.