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

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

Schematic of the Lig proteins, expression and purification of rLigB(131–645).

<p>A) The full length amino acid sequences for LigA (1224 amino acids, 128.1 kDa) and LigB (1890 amino acids, 200.8 kDa) are indicated (black line), the square boxes indicate the BIDs and the LigB C-terminal domain is shown (rectangle). The recombinant proteins used as vaccine candidates are indicated: LigB(131–645) (green boxes) includes amino acids 131–645 (53.5 kDa) and is highly identical (97.9% pairwise identity) to the same region in LigA; the LigA(631–1224), also known as LigANI, (red boxes, amino acids 631–1224, 62.8 kDa) and LigB(625–1259), also known as LigBNI, (blues boxes, amino acids 625–1259, 66.2 kDa) fragments are not highly conserved (38.1% pairwise identity). B) Expression and purification of rLigB(131–645) analysed by 10% SDS-PAGE and Coomassie staining. Lanes 1: molecular mass marker (kDa); Expression of rLigB(131–645) in an <i>E</i>. <i>coli</i>(pLigB(131–645)) clone, lane 2: supernatant (soluble) fraction and lane 3: insoluble fraction; lane 4: IMAC purified rLigB(131–645), expected molecular mass of 57.2 kDa. C) Immunoblot analysis of rLigB(131–645), following transfer the nitrocellulose membrane was probed with an anti-His-HRP antibody, lane 1: molecular mass marker (kDa); lane 2: purified rLigB(131–645).</p

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

<p>Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.</p

Pathological findings in the hamster model.

<p>Animals vaccinated with rLigB(131–645) (A, C, E and G) or the PBS control group (B, D, F and H) were euthanized 10 days PC and tissue samples were collected. Vaccinated animals showed no gross pulmonary lesions (A) or microscopic pulmonary lesions (C). Liver (E) and kidney samples (G) showed no evidence of microscopic abnormalities. Unvaccinated animals showed gross pulmonary haemorrhaging (B) and they were confirmed to be alveolar haemorrhages by microscopic analysis (D). Dystrabeculaton (loss of cohesion) of hepatocytes (F) and swelling of kidney tubular epithelial cells (H) were prominent features. (C-F, haematoxylin-eosin, 100× magnification and G-H, haematoxylin-eosin, 200× magnification).</p