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The spirochetes Borrelia burgdorferi and Leptospira interrogans are the etiological agents of Lyme disease and leptospirosis, respectively. Despite substantial biological differences among human spirochetes, all spirochetoses share a spirochetemic phase during the early stage of infection (6, 17, 20, 22). Leptospires can cause hepatitis in humans. This will result in microscopic alteration in the liver, including swelling of parenchymal cells, disruption of the liver cord, enlargement of Kupffer cells, and bile stasis in biliary canaliculi (2). In Lyme disease, liver function test abnormalities are common but mild and are most often not associated with symptoms (12). Lyme disease presenting as hepatitis and jaundice has been also reported (8). The involvement of the hepatic reticuloendothelial system (RES) in host defense by phagocytosis and killing of blood-borne spirochetes has been previously demonstrated in animals. Studies by us (18) have shown significant differences in the rat liver uptake of borreliae causing Lyme disease and of borreliae involved in relapsing fevers and have also indicated that B. burgdorferi is efficiently taken up by hepatic macrophages in the absence of serum factors. Electron microscopy studies by Faine (10) showed that in experimentally infected mice leptospires are found almost entirely in Kupffer cells and also interstitially between or in parenchymal liver cells.

The perfused liver has been used several times in the past few decades to study bacterial hepatic phagocytosis (5, 13, 15). We therefore used such a technique to evaluate the uptake and killing of leptospires in comparison with borreliae by the rat hepatic RES in the elimination of circulating bacteria. Although the mouse is a widely used model for experimental infections with borreliae, this study was performed with rats, since they are more suitable for liver perfusion. The applicability of rats for experimental studies on borreliae has also been shown previously (4, 11). It is also well known that rats are carriers of leptospires (21).

Bacterial strains, culture conditions, and labeling. The following spirochetal strains were used: B. burgdorferi IRS (ATCC 35211) and L. interrogans serovar icterohaemorrhagiae (a gift of M. Fabbi, Istituto Zooprofilattico Sperimentale, Pavia, Italy). Borreliae were cultured in Barbour-Stoenner-Kelly (BSK) II medium at 34°C, as previously reported (19), whereas leptospires were grown in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (9) at 30°C under aerobic conditions to a density of ca. 10⁸ bacteria per ml and counted in a Petroff-Hausser counting chamber. When required, bacteria were grown in the presence of 1 µCi of ¹⁴C-labeled amino acid mixture (>50 mCi/ml; Amersham Co., Amersham, United Kingdom) per ml for 96 h, washed three times with Krebs-Ringer solution (see below), counted in a Petroff-Hausser chamber, resuspended in Krebs-Ringer solution at a concentration of 1.5 × 10⁶ or 5 × 10⁷ motile organisms per ml, and used within 1 h for rat liver perfusion experiments.

Liver perfusion. Male Sprague-Dawley rats (180 to 220 g [body weight]) were used as liver donors. Food was withdrawn the evening before the experiment, and water was available ad libitum. The technique of rat liver perfusion has been already described (1) and was performed according to the method of Mortimore (16). Briefly, the animals were anesthetized intraperitoneally with pentobarbital sodium (50 mg/kg, given intraperitoneally), and the livers were perfused through the portal vein, the effluent being collected from the inferior vena cava immediately above the sovrahepatic veins. The perfusate was Krebs-Ringer bicarbonate solution containing glucose (5.55 mmol/liter), with bovine serum albumin (3% [wt/vol]) (fraction V; Sigma Chemical Co., St. Louis, Mo.). Taurocholate (sodium salt; Sigma Co.) at 0.5 mM was added to maintain the enterohepatic circulation of bile acids and bile flow. The complete blanching of all liver lobes indicated satisfactory perfusion. Oxygenation was done with O₂ + CO₂ (95/5 [vol/vol]) using Silastic tubing (Dow-Corning, Midland, Mich.). The temperature and pH of the perfusate leaving the liver were monitored throughout the experiment. The perfusate flow was established at a value of 2.2 to 2.9 ml/min/g of liver. The portal vein pressure was constant at 12-cm of water; no significant change in the levels of aspartate aminotransferase (12.0 ± 2.0 IU/liter) was observed throughout the experiment, and the pH of the effluent from the liver ranged between 7.36 and 7.42. During each perfusion, one dose of 1.5 × 10⁷ bacteria in 10 ml of Krebs-Ringer solution was infused into the portal vein. The effluent from the livers was collected at 1-min intervals over 30 min in preweighted tubes immediately after infusion. At the end of each experiment, radioactivity was measured by applying directly 100 µl of each outflow sample to cellulose acetate membrane filters (0.45-µm [pore size]) and counted after addition of Ready-Solv EP (Beckman Instruments, Inc., Fullerton, Calif.) using a Beckman LS 1801 liquid scintillation analyzer. The cumulative radioactivity throughout the experiment represented the bacteria escaping the liver uptake. A 30-min period was chosen in order to rule out bacterial efflux from the liver in later times. When required, one dose of 5 × 10⁸ labeled bacteria was infused, and spirochetes were searched for in bile by cannulation of the common bile duct with a PE-10 catheter (PE-10 Tubing; Clay Adams, Parsippany, N.J.). All animals were given humane care in compliance with institutional guidelines according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The experiments were approved by the Ethical Committee of the University of Bologna (prot.16745).

Expression of the results of liver uptake of spirochetes. The hepatic uptake was expressed as a percentage of the administered bacteria and calculated as follows: 100  the percentage outflow. The statistical analysis was performed using the Student t test for unpaired data.

Immunohistochemistry. Liver specimens, obtained at the end of the perfusions, were snap frozen in isopentane precooled in liquid nitrogen and stored at 70°C. Immunofluorescence and double-immunofluorescence techniques were performed on serial 5-mm frozen sections as previously described (3). In brief, sections were air dried 1 h and then fixed for 5 min with cold acetone, washed with phosphate-buffered saline (PBS), and incubated in a moist chamber with primary and secondary reagents for 30 min. Each incubation was followed by 3- to 5-min PBS washes. In double immunofluorescence, a 1:100-diluted convalescent-phase serum from a patient suffering from leptospirosis (the serum was a gift of M. A. Santos, INSA, Porto, Portugal) or mouse immune ascitic fluid anti-B. burgdorferi (7) was first applied, followed by rabbit immunoglobulins to human immunoglobulin G or to mouse fluorescein isothiocyanate (FITC)-conjugated immunoglobulin (Dako, Copenhagen, Denmark) diluted 1:30. Monoclonal antibodies to vimentin and desmin (Dako) were then diluted 1:20, followed by treatment with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated rabbit anti-mouse immunoglobulin (Dako), diluted 1:20 in PBS, to identify the total sinusoidal mesenchymal cell and sinusoidal stellate (Ito) cell subpopulations (3, 23), respectively. Sections were mounted in PBS-glycerol and observed with a Zeiss Axioskop microscope (Oberkochen, Germany) with epi-illumination at different wavelengths to evaluate the FITC- or TRITC-conjugated reagents.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Uptake of radiolabeled spirochetes (percentage of the infused dose) by the liver. A single dose of 1.5 × 107 14C-labeled bacteria (150,000 ± 30,000 cpm) was infused into the portal vein, and the effluent from the sovrahepatic veins was counted for radioactivity. The data shown are the means ± the standard deviations. The differences in liver uptake for L. interrogans and B. burgdorferi IRS were statistically significant (P < 0.0001).

View larger version (11K): [in this window] [in a new window]   FIG. 2.   Time course of the kinetic display of the radioactivity (means ± the standard deviations) appearing in the effluent after infusion of 1.5 × 107 labeled bacteria (150,000 ± 30,000 cpm). Symbols: , B. burgdorferi IRS; , L. interrogans.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Immunohistochemical localization of L. interrogans in rat liver sections subjected to double immunofluorescence. Thirty minutes after perfusion, the liver was fixed and liver sections were stained with human antibodies specific for leptospires followed by the addition of FITC-labeled anti-human antibodies. Afterwards, the section was probed with TRITC-labeled antivimentin monoclonal antibodies. (a) Spirochetal antigens were stained green in the cytoplasm of infected cells by FITC. (b) The same preparation shown in panel a observed at different wavelengths to evaluate TRITC reagents: the FITC-positive cells were stained red by TRITC. The results demonstrate that leptospires are associated with vimentin-positive cells.