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A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.
Leptospirosis occurs throughout the world, especially in tropical settings and developing countries.1 Both man and animal become accidental hosts through exposure to chronically infected animals, mostly rodents, that shed leptospires in their urine.2 This zoonosis is characterized by a broad spectrum of clinical manifestations, ranging from subclinical infection to Weil's syndrome3 and severe pulmonary hemorrhage syndrome.4
Similarly to human disease, leptospirosis in hamsters is acute, with lesions prominent in kidney and liver.5 The interstitial nephritis, which results in both acute and chronic kidney damage and loss of renal function as well as pulmonary hemorrhage, has been reported in hamsters, including infections with serovar Icterohaemorrhagiae and Canicola,6 serovar Pomona,7 and serovar Hardjo.8
Experimental infection with Leptospira interrogans, L. noguchii, and L. kirschneri in the hamster model usually runs a fatal course.6,9 Our group used hamsters as an experimental model for the study of leptospirosis because of the susceptibility to a broad range of pathogenic strains and reproduction of severe forms of human and animal leptospirosis.6 These results made available a panel of five virulent strains for future studies.
Reports on experimental investigation of the course of disease and histopathological findings by L. borgpetersenii serogroup Ballum in hamsters are sparse, despite the emergence of this serogroup as an important cause of disease worldwide.10–13 In this study, we carried out the additional virulence characterization in the hamster model of one strain of L. borgpetersenii serogroup Ballum, and our group reported the isolation, serogrouping, and 16S rRNA gene sequencing.14 Furthermore, this strain may now be applied in vaccine challenge experiments, comparative virulence studies, and diagnosis.
Leptospires were cultivated in liquid Ellinghausen–McCullough–Johnson–Harris (EMJH) medium (Difco, Sparks, MD) at 29°C. Density of the leptospiral challenge strain was determined by dark-field microscopy using a Petroff–Hausser counting chamber (Fisher Scientific) as described previously.15 A low passage of L. borgpetersenii serogroup Ballum strain 4E, which is a Mus musculus-derived isolate,14 was used as the leptospiral challenge strain in the mid-log phase of growth with density of ~108 cells/mL. This strain was passed two times in hamsters and stored at −70°C. The aliquots were thawed and passed in liquid medium five times before they were used as low-passage isolates in the infection experiments. Serial 10-fold dilutions in liquid EMJH medium were prepared to yield concentrations ranging from 108 to 100 cells/mL.
Male and female Golden Syrian hamsters (Biotério Central/UFPel) were used. Nine-week-old hamsters were infected with 10-fold serial dilutions ranging from 104 to 100 organisms. The number of animals for each inocula was eight according to the availability of animal facilities and the recommendation of the Committee for Animal Care and Use (UFPel). Hamsters were inoculated intraperitoneally with 1.0 mL using a 1.0-mL syringe and 22-gauge needle. All animals were monitored daily after infection. Moribund animals were euthanized and necropsied, gross lesions were observed and noted, and kidney tissue was cultured at 29°C in liquid EMJH. Furthermore, during necropsy, tissue samples from liver, kidneys, and lungs were collected and stored in neutral-buffered formalin for posterior histopathological assessment. Animals that survived infection were euthanized on the 25th day after challenge to collect tissues and determine sublethal infection. These animals were submitted to the same post-mortem assays.
Histopathology was carried out by fixing the tissue samples in paraffin and staining with hematoxilin and eosin (HE). The samples were analyzed by a pathologist that was not aware of the tissues precedence, including samples from two non-infected hamsters of the same age euthanized 9 days after inoculation with 1 mL sterile EMJH media. Fisher exact test method and GraphPad Prism 4 software systems (GraphPad Software) were used to perform the statistical analyses and generate the survival curves, respectively. Lethal dose 50% (LD50) was calculated by the recommended method.6,16
Initial clinical symptoms, such as dehydration, piloerection, tremors, and prostration, appeared around the first 4 days post-inoculation (dpi), and the mean period of death was ~10 dpi. However, females died between 9 and 14 dpi, whereas males died 8–18 dpi. The mean LD50 was 5.18 leptospires, with values of 6.81 and 4.22 for females and males, respectively. Survival curve and days to death data are shown in Figure 1.
In this study, L. borgpetersenii 4E produced lethal infection in a high percentage of male and female hamsters. The LD50 given by intraperitoneally inoculation in this experiment was less than 10 leptospires. Doses of 104 to 101 were uniformly lethal to male hamsters, whereas one female hamster that received a dose of 101 leptospires survived. All animals in the 100 group survived to 25 dpi (Figure 1) and did not show any clinical abnormalities.
The gross findings at necropsy were that hamsters infected with L. borgpetersenii showed jaundice, pulmonary hemorrhages, and a dark congestive kidney process with varied intensity according to the inoculums group (full macroscopic analyses not shown). Histopathological lesions detected in the inoculated hamsters also depended on the inoculums group. Table 1 summarizes the main findings in the target organs (kidney, liver, and lung). Animals that sustained high-challenge inoculations (103 and 104) had less chances of developing extensive macroscopically observable pulmonary hemorrhage than the animals that sustained low-challenge doses (101 and 102; P < 0.01). Likewise, animals that were inoculated with high doses seemed more subjected to severe jaundice than others (P < 0.01). When microscopic lesions were compared, no statistical difference was observed.
Virulence characterization assays and LD50 determination are important to further study leptospirosis, especially with vaccine trials. There is a great need to develop cross-protective leptospirosis vaccines17,18 that may retire current bacterins. To assess cross-protection, standardized strains of different species and serogroups of Leptospira are needed, and few are available.19–23 This work attempts to fill this void by describing the pathogenesis and LD50 of a Leptospira isolate, which is not only highly virulent but also belongs to a serogroup of growing importance in recent years.10–13
Experimental reproduction of leptospirosis was successfully achieved in the hamster model using strain 4E. Furthermore, we describe a thorough standardization of the LD50 and pathological inflictions. In 2008, Silva and others6 published a similar study using different strains that enabled the execution of several assays, mainly vaccine trials24 and pathogenicity studies.25 The current work, associated with the complete genome sequence of L. borgpetersenii serovar Hardjo,26 should allow for the development of studies related to heterologous vaccine trials as well as compare pathogenesis with virulence studies.
The mean LD50 was of 5.18 leptospires. Compared with previous studies, this strain is similarly virulent to L. interrogans serovar Canicola strain Kito (2.7 leptospires) and L. noguchii serovar Autumnalis strain Bonito (3 leptospires).6 It is substantially more virulent than L. interrogans strain Fiocruz L1-130 (~80 leptospires), which was widely used because of the availability of the complete genome sequence.27 Highly virulent strains with low LD50 are desirable for simulating the disease in several studies, because it has been argued that high-challenge doses make poor experimental models.2
Strain 4E produced most of the classical clinical and pathological signs of leptospirosis, including but not limited to pulmonary hemorrhages, jaundice, and dark congestive kidneys. Microscopically, it produced mostly classical lesions like those described in naturally infected humans and animals.2,28 Furthermore, it was observed in the previous study14 that strain 4E produced pulmonary hemorrhage, which was associated with leptospirosis acute pulmonary syndrome, the most severe form of leptospirosis.4
Another interesting result was the statistically different pathological findings in the comparison among high- and low-dose challenge subjects. Animals that died early (high-challenge dose) had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01) than animals that died late (low-challenge doses) in the experiment. This may indicate that the lungs are compromised later than the kidneys and/or liver when the disease occurs, but high-dose challenge kills before such lesions occur, adding to the claim that high-challenge doses make poor experimental models.2 The strain was deposited in the Fiocruz/BA collection under the responsibility of Albert Ko, and it is available to the scientific community.
In conclusion, this work successfully describes the LD50 of L. borgpetersenii serogroup Ballum strain 4E. Furthermore, pathological findings revealed additional insights into the pathology of the disease. This strain is now available to be used in additional studies, especially vaccine trials and cross-protection assays.
The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for the financial support and sponsorship of the authors. We are grateful to the staff of the Biotério Central of Universidade Federal de Pelotas for their technical support.
Financial support: This work was supported by CNPq.
Authors' addresses: Juliana Alcoforado Diniz, Samuel Rodrigues Félix, Amilton Clair Pinto Seixas Neto, Flávia Aleixo Vasconcellos, André Alex Grassmann, Odir Antônio Dellagostin, José Antonio Guimarães Aleixo, and Éverton Fagonde da Silva, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil, E-mails: rb.moc.oohay@zinida_ujuj, moc.liamg@frleumas, moc.liamg@saxiesnotlima, rb.moc.oohay@savelaue, email@example.com, rb.ude.lepfu@rido, rb.ehct.lepfu@agajtoib, and rb.ude.lepfu@avlisfe. Josiane Bonel-Raposo, Faculdade de Veterinária, Universidade Federal de Pelotas, Pelotas, RS, Brazil, E-mail: moc.liamtoh@osopar-lenobj.