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Establishment of a leptospirosis model in guinea pigs using an epicutaneous inoculations route
- Yan Zhang†1,
- Xiao-Li Lou†1, 2,
- Hong-Liang Yang3,
- Xiao-Kui Guo1,
- Xiang-Yan Zhang1,
- Ping He1Email author and
- Xu-Cheng Jiang4Email author
© Zhang et al; licensee BioMed Central Ltd. 2012
Received: 16 August 2011
Accepted: 25 January 2012
Published: 25 January 2012
Leptospires are presumed to enter their host via small abrasions or breaches of the skin. The intraperitoneal route, although commonly used in guinea pig and hamster models of leptospirosis, does not reflect conditions encountered during natural infection. The aim of this study is to develop a novel leptospirosis guinea pig model through epicutaneous route and to elucidate the pathogenesis of leptospirosis in experimental guinea pigs by comparing the data from other studies using different infection routes.
The guinea pigs were inoculated with 5 × 108 Leptospira interrogans strain Lai onto either shaved-only or abraded skin. The guinea pigs were sacrificed at 2, 8, 24, 48, 72, 96 and 144 h post-infection (p.i.) followed by harvest of the lungs, liver, kidneys, spleen, and the skin around the inoculated sites for further examinations. Hematoxylin and eosin (HE) staining and electron microscopy were used to detect the pathologic changes. Real time PCR and immunohistochemistry staining were performed to detect dynamic distribution of leptospires in blood and tissues, respectively.
In the guinea pigs with abraded skin inoculations, leptospires were detected in blood as early as 2 h post infection (p.i.) and then disseminated to the liver, lungs and kidneys of almost all animals within 96 h p.i.. Leptospires were also detected engulfed in the swelling vascular endothelial cells and were frequently aggregated around the capillaries in the dermis and subcutaneous tissue under the inoculated site. For the guinea pigs with abraded skin inoculations, hemorrhage at the dermis around the inoculated site was found before the appearance of internal organs hemorrhage, severe lesions such as hemorrhages in the lungs, nephritis, jaundice, haematuria were also observed, and two of seven guinea pigs died at 144 h p.i. while no lesions and leptospires were detected in the shaved-only guinea pigs using the same dose of strain Lai.
Intact keratinocyte layer is a very efficient barrier against leptospires, and intact skin can prevent the infiltration of leptosipres to the host. Leptospires can penetrate abraded skin and quickly establish a systemic infection by crossing tissue barriers. We have successfully established a novel leptospirosis guinea pig model through epicutaneous inoculations route, which replicates a natural course of infection and appears to be an alternative way to investigate the pathogenesis of leptospirosis, especially in terms of early stage of host-pathogen interactions. This novel model may also be advantageous for studies of the mechanisms involved in cutaneous barriers and epidermal interactions with this organism.
Leptospirosis is a worldwide bacterial zoonosis caused by several species of invasive spirochetes belonging to the genus Leptospira. It affects humans in both rural and urban areas, particularly in developing countries with warm and humid climate [1–3]. Water contaminated by urine from animal reservoirs is the main source of human infection, usually through cut or abraded skin. Leptospirosis is characterized by a broad spectrum of clinical manifestations, ranging from subclinical infection to Weil's syndrome, a severe and potentially fatal disease characterized by hemorrhage, acute renal failure and jaundice . Deaths may occur in less than 72 h after the advent of respiratory signs and symptoms such as severe hemorrhage of lungs, which usually appear between the fourth and the sixth day of disease .
The use of experimental models remains a critical component for elucidating pathogenesis of leptospirosis. Young guinea pigs and hamsters are the most commonly used experimental models for acute leptospirosis . The intraperitoneal (i.p.) inoculation route is the most widely applied infection route by producing a lethal infection in experimental animals and mimicking the clinical symptoms of severe leptospirosis in humans [7–10]. However, this route of infection does not reflect real conditions encountered during natural infection, because leptospires are believed to enter the host via mucous membranes or abrasions of the skin. It has been a long time for researchers to challenge animals through alternative routes to mimic natural entry of leptospires into hosts. Even about one century ago, Ido and his colleagues attempted to reproduce natural conditions by conveying the leptospries directly to the guinea pig by the bite of rat (carrier of leptospires). The results indicated that leptospirosis is rarely transmitted by the bite of rat . Since then, different infection routes such as conjunctival (c.j.) and subcutaneous (s.c.) have been employed in canine, horse, hamster and guinea pig, and resulting in acute leptospirosis in inoculated animals [11–16]. By using infection routes different from the classic i.p. inoculation, these studies contributed to the elucidation of pathogenesis of leptospirosis in experimental animals. However all these methods bypassed the epidermis of host, the entry route and mode of leptospires directly via epidermis have been poorly studied as of today. There is still little data on how the leptospires interact with the epidermis and if the inoculated leptospires can penetrate skin and disseminate in host subsequently.
In this study, we examined the ability of leptospires to produce infections in guinea pigs when applied to damaged or undamaged skin. The results showed that infection with virulent leptospires, using abraded skin inoculation route of infection, produced typical leptospirosis in guinea pigs, whereas there were no symptoms in guinea pigs through shaved-only skin. The availability of this novel model will enable understanding of the pathogenesis of leptospirosis, as well as to study cutaneous barriers and epidermal interactions with this organism.
Leptospiral strain and growth conditions
The L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was obtained from the Institute for Infectious Disease Control and Prevention, Beijing, China. Virulence of leptospires was maintained by iterative passage in guinea pigs. Leptospires were grown in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium [17–19] at 28°C under aerobic conditions to the mid-log-phase and were counted using Petroff-Hausser counting chamber.
Young male health guinea pigs, weighting 150-200 g each, were purchased from Institute of Biological Products of Shanghai. All guinea pigs were housed in specific cages containing autoclaved bedding, sterilized feed and water. The animal experiments were approved by the Animal Research Committee of Shanghai Jiao Tong University School of Medicine.
Epicutaneous inoculation of L. interrogans on the flank skin of guinea pigs was performed using the modified procedure as previously described . Briefly, the guinea pigs were carefully shaved over their left flank one day before inoculation, and then disinfected with iodine, washed with 75% alcohol followed by saline. The abraded skin was then prepared by gentle scraping with a surgical scalpel blade until a non-bloody glistening skin layer resulted, representing damage to the stratum corneum water barrier. An inoculum of 5 × 108 leptospires in 20 μl of sterile saline was added to 4 × 4 mm filter discs (Thermo Fisher Scientific, USA). The filter discs were then added on the either shaved-only or abraded skin. Sterile saline of 20 μl was applied on the abraded skin as negative control. Inoculated sites were covered with a 1.0 cm2 piece of plastic sheet and overwrapped with Band-Aid waterproof tape (Johnson, USA). At different time points after inoculation, the number of leptospires left on the filter discs were checked by extensively PBS washing and then counted using Petroff-Hausser counting chamber. At 2 h post-infection (p.i.), there were about 5 × 104 leptospires left on the filter discs, suggesting that only one ten-thousandth of inoculums (5 × 104/5 × 108 leptospires) have not penetrated to the skin. There was no leptospires detected after 24 h p.i.. It was supposed that all the leptospires were inoculated on the skin/or abrade skin at 24 h p.i.. Because guinea pigs were sacrificed at previously determined time points, the filter discs were removed after 2-24 h. Generally seven of leptospires infected abraded-skin guinea pigs, three of leptospires infected shaved-only guinea pigs and three of negative controls per time point were used in 3 independent experiments.
Monitoring of infections
The guinea pigs were euthanized at 2, 8, 24, 48, 72, 96 and 144 h p.i.. Blood samples were collected by cardiac puncture for quantitative real-time PCR. The lungs, liver, kidneys, spleen, and the skin around the inoculated sites were harvested for histologic examinations. Paraffin sections were prepared and then stained with hematoxylin and eosin (HE). The rabbit antiserum specific to L. interrogans strain Lai was prepared in our lab using a modified procedure as previously described . Immunohistochemistry staining was performed using the EnVision™ system (EnVision system, Dako, USA) . In brief, paraffin-embedded tissue sections were dewaxed and rehydrated, treated with 3% H2O2 in methanol for 10 min, and then incubated in 0.1% trypsin at 37°C for 30 min. Sections were incubated in primary rabbit antibody (1:6000 dilution) specific for L. interrogans strain Lai for 12 h at 4°C, followed by EnVision™ for 30 min, then visualized with 3,3'-diaminobenzidine (DAB), and counter-stained with modified hematoxylin. Tissues for ultrastructural studies using a modified procedure as previously described . Briefly, tissues around the wound were removed and fixed in 2% glutaraldehyde immediately for 24 h, then post-fixed in 1% osmium tetroxide, dehydrated in graded ethanols, and embedded in Epon 618. Ultrathin (70 nm) sections were stained with uranyl acetate and lead citrate, and examined with a PHILIP CM-120 electron microscope.
Blood DNA extraction and the following real-time PCR
Genomic DNA of leptosipres from blood samples was extracted using a blood DNA purification kit according to manufacturer's instruction (Omega, USA). The concentration of leptospires in animal blood was quantified by real-time PCR using Applied Biosystems 7500Fast. All reactions were performed with the Power SYBR Green PCR Master Mix (Applied Biosystems, USA). The 116 bp 16S rRNA gene amplicons of leptospires were quantified using the primers F (5'-TCC TGG CTC AGA ACT AAC GC-3'), and R (5'-TCC CAG ACT CAG AGG AAG AT-3'). PCR conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of amplification 95°C for 15 s and 60°C for 60 s. The standard curve for quantification was made using a modified procedure as previously described . In brief, the 116 bp 16S rRNA gene amplicons were cloned into the vector pMD19 by using the TA cloning kit (Invitrogen, USA). There are two copies of the 16S rRNA gene per Leptospira. The 10-fold dilutions of recombinant plasmid with 16S rRNA was used to establish the standard curve for quantification. Results were expressed as the number of leptospires in 1 μl blood. The real-time PCR was performed in duplicate for each DNA extraction. Three guinea pigs were used for each time point in each group.
Occurrence of hemorrhage after inoculation
In contrast with progressing and severe leptospirosis in guinea pigs with the abraded skin leptospires inoculation, none of these features was observed in the shaved-only and saline inoculated control animals (Figure 1 and 2).
Leptospires distribution in blood and tissues of infected guinea pigs
Intraperitoneal injection is the most widely used infection route in experimental leptospirosis studies [9, 24]. It reproduces the processes of the human leptospirosis in the animal models using a very easy way. However, intraperitoneal injection does not reflect the natural transmission of the pathogen. Leptospires are thought to enter the human body via cuts or abrasions in the skin. The entry of leptospires directly via epidermis has been poorly studied. Some reports of clinical leptospirosis cases have clearly identified the initial cutaneous injury , others have not noted such a preexistent lesion [26, 27]. It is not known whether the organism can penetrate intact skin or abraded skin. In this study, we established a guinea pigs leptospirosis model using epicutaneous inoculations route, to gain a better understanding of host-pathogen interaction and the pathogenesis of leptospirosis.
In this study, guinea pigs were inoculated with leptospires onto either shaved-only or abraded skin. Guinea pigs with abraded skin displayed clinical signs of leptospirosis. In contrast, lesions were not detected in the shaved-only animals which were inoculated the same amount of virulent L. interrogans strain Lai. These data confirmed that the intact keratinocyte layer is a very efficient barrier against leptospires, and intact skin can prevent the infiltration of leptosipres to the host.
It should be noted that the L. interrogans strain Lai used in this research was originated from a female patient who died of pulmonary hemorrhage after an infection with this organism, which had previously been studied in a variety of animal models and found to develop a typical leptospirosis in guinea pigs with intraperitoneal route [9, 28, 29]. The inoculation dose was referred to the previous guinea pig model reported in our lab .
Our study here showed that infection with the L. interrogans strain Lai using abraded skin inoculation route of infection produced a lethal infection in guinea pigs that mimicked the clinical characteristics of severe leptospirosis in patients, as described elsewhere [5, 30, 31]. The main clinical signs were serious pulmonary hemorrhage, jaundice, retroperitoneal hemorrhage and renal hemorrhage.
Our data showed that virulent leptospires can rapidly (within 2 h) penetrate the abraded epidermis and enter the dermis; at some point within 2 h p.i., the invading organisms also distribute to blood. Attachment to host cells and host extracellular matrix (ECM) components is likely the necessary step for leptospires to penetrate, disseminate and persist in mammalian host tissues. Consistent with the ability of L. interrogans to migrate through host tissues, a wide range of adhesion molecules were discovered in these organisms that may facilitate this process [32–34]. It has been reported that many leptospiral proteins, including LigA/B, Lsa21, Lsa27, LenA to F, LipL32, OmpL37, TlyC and LipL53, have affinity for ECM and cell surface in vitro [32–41]. Some of these proteins, such as OmpL37, were reported to have the strong binding affinity for skin and aorta elastin, and might facilitate the attachment of leptospires to elastin-rich inner layer of the skin as well as vascular structures .
It is evident that leptospires penetrate abraded skin and quickly establish a systemic infection by crossing tissue barriers. It is likely that L. interrogans can move through the tissue barrier by association with blood vessels, because leptospires were detected aggregated around the capillaries in muscular layer and peritonaeum in this study. It was thought that leptospires, like other spirochaetes, spread through intercellular junctions . However, they have been shown to efficiently enter host cells in vitro [43–45]. Previous work accomplished by Martinea-Lopez et al. demonstrated that L. interrogans can disrupt the dynamics of the actin cytoskeleton in the human microvascular endothelial cell line and rapidly translocate through the cell layers . Other studies in human leptospirosis have shown that leptospiral antigens were detected in the cytoplasm of the endothelial cells of septal capillaries . Consistent with these results, leptospires were detected intracellularity in the vascular endothelial cells and disruptions of vascular basement membrane were also observed in this study. Our findings suggested that leptospires cross endothelial barrier and cause heamatogenous dissemination by pass through the endothelial cell cytoplasm.
When L. interrogans strain Lai was inoculated on the abraded skin, localized changes around the inoculated site were detected. All of the guinea pigs showed hemorrhage at the dermis around the site-inoculation before the appearance of internal organs hemorrhage. Skin hemorrhage was rarely reported in animals infected experimentally through the i.p. route, and little attention has been called for. The mechanism of hemorrhage caused by leptospirosis has not been elucidated yet. Factors contributing to the hemorrhage might involve direct action of toxins and autoimmune process. Nicodemo and coworkers detected the intact leptospires in capillary endothelial cells, indicating the lung injury is directly triggered by leptospires and/or by their toxic products . Another study demonstrated the deposition of antibodies and complement along the alveolar basement membrane of infected guinea pigs, indicating pulmonary hemorrhage might be led by autoimmune process . Our data showed that abundant leptospires were detected in the dermis and subcutaneous tissue of hemorrhagic area and were rarely detected in adjacent none hemorrhagic areas, confirming the high burden of leptospires in the dermis is an important factor to cause hemorrhage. Humoral immune response seems not be associated with the pathogenesis of skin hemorrhage, as dermis hemorrhage developed as early as 8-24 h p.i.. Further examination of the local hemorrhage may give a clue to understand the mechanism of hemorrhage in this disease.
Hemorrhage in the skin is produced as one of the general symptoms in clinical cases . However, Hemorrhage localized at infected site was rarely recognized clinically. Local hemorrhage in our experiment model might caused by high dose inoculation of leptospires. When in nature infection, it seems like that low dose leptospires in the cuts or abrade skin will not cause skin hemorrhage until large amount of pathogen proliferated in the circulation, and then extensive skin hemorrhage will be produced.
Recently, Lourdault and his colleagues compared different routes (i.p., c.j. and s.c. inoculation) of infection and the dissemination of leptospries in blood and tissues of guinea pigs using multiple methods including real-time PCR . The results showed infected guinea pigs developed similar physical signs and pathological changes after i.p., s.c. and c.j. inoculation with leptospires, and the bacterial burden in tissues and histopathology revealed no major differences between the three routes of infections . In the guinea pigs with abraded skin inoculation, our real-time PCR results showed that the bacteraemia peaked at 96 h p.i. and then quickly decreased at 144 h p.i., which were consistent with the result of i.p. inoculated guinea pigs or s.c. inoculated hamsters reported by Lourdault and Truccolo respectively [15, 16]. It is interesting to note that the high leptospires burden (3 × 105 leptospires ml-1) detected in the blood at 2 h p.i., and then quickly dropped by 1 log at 8 h p.i.. It is speculated that high dose (5 × 108) leptospires inoculation cause a rapid flood of leptospira from the inoculated site to the bloodstream, then the majority of the leptospires were cleared by the innate immune system in the following several hours. As pathogenic Leptospira were reported to be able to survive, and be more resistant to the action of the complement system [47–49], polymorphonuclear neutrophils (PMNs), which constitute the largest population of intravascular phagocytes, are expected to play an important role in leptospiral clearance. It was reported that PMNs are able to kill pathogenic strains of Leptospira by oxygen dependent and independent mechanisms . However, some experimental models showed that phagocytosis of pathogenic Leptospira by neutrophils and macrophages is only effective if this pathogen is opsonized by specific IgG [51–53]. Further investigations on PMNs activation and elimination of pathogenic leptospires are required to elucidate the establishment of innate immune responses in leptospirosis.
The traditional intraperitoneal inoculation is easy to handle and allows reproducible amounts of leptospires to be introduced. It is still the most widely used model to study the systemically infection of leptospirosis. However, there are some shortages of i.p. or other non-epicutaneous routes when apply on the pathogens causing infection through skin. In study performed by Bischof and colleagues, the subcutaneous injection of B. anthracis (Sterne strain, which lacks the pX02 capsule plasmid) caused lethal infection in C57BL/6 mice, while quite resistant to epicutaneous inoculation of B. anthracis onto abraded skin . This study suggested that our epicutaneous inoculation model would be an alternative way to apply the characterizations of Leptospira mutants that are deficient in protein with binding affinity for skin.
In summary, our current research demonstrated L. interrogans strain Lai had the ability to penetrate lesional epidermis after epicutaneous inoculation in guinea pigs, and also had the ability to disseminate systemically from the skin within 48 h of such inoculation. The guinea pigs leptospirosis model with an epicutaneous inoculation route described here replicated a natural course of infection and revealed epicutaneous inoculation might be an alternative route to investigate the pathogenesis of leptospirosis, especially when focus on the early steps of infection while the intraperitoneal inoculation is still a classic and main infection route due to its easy to handle feature. Our current model may also contribute to gain a better understanding of the mechanisms involved in cutaneous barriers and epidermal interactions with this organism, and consequently a delineation of the host-bacterium relationship with the aim of establishing prevention, early diagnosis, and efficient therapeutic regimens.
This work was supported in part by the National Natural Science Foundation of China (grant numbers 30770820, 30970125, 81101264 and 81171587), the National Key Program for Infectious Diseases of China (grant2009ZX10004-712), the Program of Shanghai Research and Development (10JC1408200), and the research project of Shanghai Municipal Health Bureau (grant number 2008045).
We thank Bao-Yu Hu (Department of Medical Microbiology and Parasitology, Shanghai Jiao Tong University School of Medicine), Yi-Xin Nie (National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention) for help in bacterial culture preparation. We are thankful to Dr. Jin-Hong Qin for thoughtful comments on the manuscript.
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