Increasing interest in the development of a recombinant leptospirosis vaccine has emerged in the past few years. The field was further stimulated by the whole genome sequencing of four strains from two pathogenic Leptospira
species. Promising results have been reported by the use of recombinant proteins in hamster [13
]. In addition, naked DNA and adenovirus used as a vaccine vector for a leptospiral antigen have also been evaluated in a gerbil model [18
]. In these studies, the ability of sub-unit vaccine candidates to induce immunoprotection against heterologous serovars was not evaluated. However, a recent study reported heterologous protection in a gerbil model using plasmids encoding LipL32 (Hap1) from serovars Autumnalis and Grippotyphosa. Immunization with this construct conferred protection against L. interrogans
serovar Canicola challenge [16
]. A more complete evaluation of the capacity of vaccine candidates to induce cross-protective response requires a well-characterized panel of virulent strains representing serovars of public health and veterinary importance. We report here a suitable model for evaluation of vaccine candidates against a wider range of pathogenic serovars.
In our study, we performed challenge experiments in hamster model with five highly virulent Leptospira
strains. Mice and hamster models have often required inocula as high as 106
leptospires to induce death [15
]. Despite high doses, some strains are unable to induce mortality in all control animals. For example, challenge with a 108
inoculum of Pomona strain induced death in 25-57% of unvaccinated hamsters [15
], which in turn complicates the conclusions on the effectivenes of a vaccine candidate. The need for high challenge doses of up to 106
organisms to produce lethal infection may be due to the non-susceptibility of the animal model (mouse) [17
] or low virulence of the strains. Protective effects would be better evaluated using strains with an LD50
in a susceptible host, such as the hamster. In a previous evaluation of homologous protection using Fiocruz L1-130 strain, fragments of leptospiral immunoglobulin-like proteins prevented lethal disease in hamsters while unvaccinated controls showed a high rate of lethality (100% of 76 animals infected with 103
Copenhageni Fiocruz L1-130 strain was isolated from a patient identified during the flood-associated outbreak of leptospirosis in Salvador, Brazil, in 1996 [11
]. This isolate was also the target of a multicenter initiative to sequence its genome [8
]. Additionally, it has been used for studies on pathogenesis and leptospiral proteomics [20
]. Serovar Copenhageni is the most common leptospiral isolate in Salvador and also in other urban areas of Brazil [10
] and its low LD50
demonstrates that it is a highly virulent pathogen for hamsters, which has been used in previous immunoprotection experiments [14
serovar Canicola is the second most important agent for urban leptospirosis in Brazil [10
]. In our study, we could demonstrate it as a highly virulent strain for hamsters with a general LD50
of 2.57 leptospires. Serovar Canicola may cause severe disease in animals and has already been associated to a pulmonary hemorrhage human leptospirosis outbreak in rural areas of Nicaragua [37
is a pathogenic species present in the American continent and has been recently reported in the South of Brazil [38
]. It had been previously isolated from human, armadillo, toad, spiny rat, opossum, nutria, Mustela nivalis
, cattle and sheep, showing a wide variety of domestic and wild hosts [2
]. Strains Bonito and Cascata were isolated from human patients with clinical leptospirosis, while Hook strain was isolated from a stray dog with lethal leptospirosis. All strains were virulent for hamsters.
These human and animal virulent isolates were submitted to hamster passages before their use in LD50 experiments. Only strains Isoton and Skoll, which did not cause lethal disease and could not be recovered from hamster kidneys inoculated with 108 leptospiras, were not further evaluated. It is conceivable that the observed high virulence in LD50 experiments reflects a virulence artificially obtained by successive hamster passages (“hamster adaptation”), and not the natural virulence of each isolate. However, our goal in this study was to select distinct virulent strains to be used in hamster model regardless of the original virulence characteristics of the isolates.
The lack of virulence of strain Isoton, an isolate from the blood culture of a patient with severe leptospirosis might be explained by the successive in vitro
propagation: 31 passages may have allowed mutations to occur rendering the strain avirulent. It has already been reported that high-passage strains can present impaired virulence [40
]. On the other hand, although uncommon, some strains may be virulent in host animals but not in the experimental model as is the case for serovar Hardjo subtype A strains [7
]. The Skoll strain was isolated from an asymptomatic mouse. In this case it is accepted that avirulent leptospires may colonize renal tissues of mammalian hosts [40
]. Similarly, we have previously isolated the L. noguchii
strain Caco from an asymptomatic sheep that failed to cause disease in the hamster model [38
]. The lack of virulence in these strains precludes their use in immunoprotection experiments.
The hamster model reproduced pathologic findings observed in acute lethal forms of human and experimental leptospirosis, as previously described [42
]. The presence of microscopic pulmonary hemorrhage in the absence of gross features has also been reported in humans and in guinea pigs [25
]. The pattern of acute cell swelling in fulminant disease and a picture of multifocal regeneration tubular foci and interstitial nephritis in more prolonged illness have also been reported in humans [42
]. Taken together, the reproducibility of acute lethal infection and target organ pathology makes the hamster model suitable for standardization of virulent strains and immunoprotection assays.
In conclusion, we characterized the virulence of five clinical isolates of Leptospira belonging to five different serogroups. These highly virulent strains are currently been used in experiments aiming at evaluating homologous and heterologous protection induced by killed whole-cell and recombinant vaccine candidates against acute lethal leptospirosis in the hamster model.