American Journal of Pathology, Vol. 141, No. 5, November 1992 Copyright © American Association of Pathologist
Cystitis Induced by Infection with the Lyme Disease Spirochete, Borrelia burgdorferi, in Mice Stephanie Czub,* Paul H. Duray,t Rex E. Thomas,* and Tom G. Schwan* From the Department of Health and Human Services,* Public Health Service, National Institutes ofHealth, National Institute ofAllergy and Infectious Diseases, Laboratory of Vectors and Pathogens, Arthropod-borne Diseases Section, Hamilton, Montana, and Fox Chase Cancer Center,t
Philadelphia, Pennsylvania
Previous studies have demonstrated that the urinary bladder is a consistent source for isolating the Lyme disease spirochete, Borrelia burgdorferi,from both experimentally infected and naturally exposed rodents. We examined histopathologic changes in the urinary bladder of different types of rodents experimentally infected with Lyme spirochetes, including BALBIc mice (Mus musculus), nude mice (M. musculus), white-footed mice (Peromyscus leucopus), and grasshopper mice (Onychomys leucogaster). Animals were inoculated intraperitoneally, subcutaneously, or intranasally with low-passaged spirochetes, highpassaged spirochetes,; or phosphate-buffered saline. At various times after inoculation, animals were killed and approximately one-half of each urinary bladder and kidney were cultured separately in BSKII medium while the other half of each organ was prepared for histologic examination. Spirochetes were cultured from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes while we were unable to isolate spirochetes from any kidneys of the same mice. The pathologic changes observed most frequently in the urinary bladder of the infected mice were the presence of lymphoid aggregates, vascular changes, including an increase in the number of vessels and thickening of the vessel walls, and perivascular infiltrates. Our results demonstrate that nearly all individuals (93%) of the four types of mice e-xamined had a cystitis associated with spiro-
chetal infection. (AmjPathol 1992, 141:1173-1179)
Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne spirochete infecting humans and a variety of both domestic and wild animals throughout
many regions of the world.1-3 In the hyperendemic regions of the northem midwest and northeastern United States, the white-footed mouse, Peromyscus leucopus, is the primary reservoir for the spirochete and an important host for immature Ixodes dammini ticks that transmit the spirochete.4'5 Lyme disease in humans is a multisystem disorder that varies tremendously in its clinical presenta-
tion.2'S
Numerous reports have demonstrated the usefulness of various rodents as experimental animals for investigating the infectiousness of B. burgdorferi,9-13 to examine specific aspects of the pathogenesis of Lyme disease,1618 to determine in vivo antibiotic susceptibility,19 and to examine potential vaccines. 2021 Studies with animals and reports of urine of humans suggest that either the urinary bladder or the urinary tract may be infected with the Lyme spirochete. Previous investigations have shown that B. burgdorferi may be present in the urine of naturally infected white-footed mice,22 and that the urinary bladder of experimentally infected and naturally infected rodents often produces spirochetes when this organ is triturated and inoculated into BSK-11 medium.10'23'24 Antigens of B. burgdorferi have been detected in the urine of both laboratory mice experimentally infected with B. burgdorferi and humans with Lyme disease,25, "6and B. burgdorferi has been demonstrated in the bladder wall of white-footed mice by direct immunofluorescence staining.10 As well, the amplification of B. burgdorferi DNA using the polymerase chain reaction has been used recently to detect spirochetes in the urine of humans with Lyme disease,27 and bladder function disturbance leading to urine retention has also been reported.2e The frequent infection of spirochetes in the uriDr. Czub's current address is the Institute of Pathology, University of
Wurzburg, Germany. Dr. Duray's current address is the Department of Pathology, Brgham and Women's Hospital, Harvard Medical School, Boston, Massachusetts. Dr. Thomas' current address is Paravax, Inc., Fort Collins, Colorado. Accepted for publication May 14, 1992. Address reprint requests to Dr. Tom G. Schwan, Department of Health and Human Services, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Vectors and Pathogens, Arthropod-bome Diseases Section, Hamilton, MT 59840.
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nary bladder of experimental and wild rodents has raised the question as to whether some cases of interstitial cystitis in humans might be caused by B. burgdorferi.'9 Because of the previous studies cited above, demonstrating that the urinary bladder of rodents often produces isolations of B. burgdorferi, including mice that have been infected for 16 months,30 the objective of the current study was to determine if infection by Lyme spirochetes resulted in any histopathologic changes in this organ.
Materials and Methods
Animals and Housing Conditions The following strains and species of mice were infected with B. burgdorferi: laboratory mice (Mus musculus, RML BALB/c/Ann), grasshopper mice (Onychomys Ieucogaster), white-footed mice, and nude Harlan SpragueDawley mice (HSD nu/nu, M. musculus). All animals were obtained from the Rocky Mountain Laboratories animal production facility. Both sexes were used, and all animals were 1 to 3 months old when inoculated. RML and HSD nu/nu mice were kept in groups of four to five animals per cage. The grasshopper mice and white-footed mice were kept separately with only one animal per cage.
Strains of B. burgdorferi and Animal Inoculation Borrelia burgdorferi strains Sh-2-82, CA-2-87, and ECMNY-86 were described elsewhere.10 Briefly, Sh-2-82 originated from Ixodes dammini ticks from Shelter Island, New York; CA-2-87 originated from pacificus ticks from Tulare County, California; ECM-NY-86 originated from a skin biopsy from a EM lesion of a human patient in Southampton, New York. Fresh, low-passaged cultures (passage 5) of Sh-2-82 and ECM-NY-86 were prepared in BSK-11 medium,31 removed from the medium by centrifugation, and resuspended in PBS-5 mmol/l MgC12 (pH 7.4), and counted in a Petroff-Hauser bacteria counting chamber. A higher-passaged (passage 15) noninfectious culture of CA-2-87 was prepared in the same manner. Animals infected with the low-passaged strain were inoculated by one of three routes including intraperitoneal, subcutaneous, or intranasal inoculation with approximately 7 x 107 B. burgdorferi in PBS-5 mmol/l MgC12 (pH 7.4). Animals inoculated with the high-passaged strain were injected intraperitoneally with the same number of spirochetes and caged separately from mice inoculated with low-passaged spirochetes. Six mice (two of each species) were also inoculated with PBS intraperitoneally for negative controls and kept in separate cages in
the same room. The animals were examined twice per week for their health status. Between 14 days and 8 months postinfection, the mice were anesthetized with ether, killed, and the kidney and urinary bladder were removed under sterile conditions. Ten uninoculated nude mice were also examined.
Cultivation of Borrelia burgdorferi One half of each kidney and urinary bladder was used for the histologic examination (see below), and the other half was used to isolate spirochetes as described previously.10 Briefly, each tissue was triturated in 1 ml BSK-11 medium in a glass tissue grinder. Half of this suspension was inoculated into a tube containing 9 ml plain BSK-ll medium, and the other half into a tube containing 9 ml BSK-1l medium plus phosphomycin (100 ,ug/ml) and rifampin (50 ,ug/ml). The cultures were incubated at 340C and checked by darkfield microscopy every few days for 2 to 3 weeks. Organs of control animals were treated in the same way. Bladders from the 10 uninoculated nude mice were only examined histologically.
Tissue Processing Imprint smears of one half of the urinary bladder and the left kidney from infected and normal animals were fixed with methanol and stained by a direct immunofluorescence assay using rabbit anti-B. burgdorferi antibodies labeled with fluorescein isothiocyanate (FITC). Afterwards, the organs were prepared for sectioning using standard methods, cut in 4-,. serial sections and mounted on slides, and examined by light microscopy.
Staining Procedures Hematoxylin and eosin (H&E) stain was used on all slides. Methyl-green-pyronin stain (Sigma) was used to detect plasma cells and mast cells. Immunohistochemical stains (APAAP, ABC peroxidase), direct immunofluorescence with rabbit anti-B. burgdorferi antibody labeled with FITC, and indirect immunofluorescence with monoclonal anti-B. burgdorferi outer surface protein A antibody H5332,32 monoclonal anti-flagellin antibody H9724,33 and polyclonal rabbit anti-B. burgdorferi antibody were used to detect B. burgdorferi in the tissues.
Analysis of Histologic Changes in the Urinary Bladder Sections from the urinary bladder of 48 mice were examined for histologic abnormalities, including 29 from mice
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inoculated intraperitoneally with infectious spirochetes, three from mice inoculated intraperitoneally with noninfectious spirochetes, six from mice inoculated with only PBS, and 10 uninoculated nude mice. Part of the same bladder from all the mice (excluding the 10 nude control mice) were also cultured in BSK-ll. The identity of the mice and the results of cultivation were not known when the slides were examined and the histologic changes quantified. Pathologic changes in the bladder included the number and size of lymphoid aggregates, changes in vasculature, and alterations in bladder tissues. Tissues were scored to quantify the various changes and included the following: the number of lymphoid aggregates per section varied from 0, 1, 2, 3, 4, or more and received scores of 0 to 5, respectively. Sizes of the aggregates measured with an ocular micrometer for relative size to one highpower field (HPF, 400x magnification), varied from less than 25%, 25% to 50%, 50% to 75%, 75% to 100%, and greater than 100% of HPF and received scores of 1 to 5, respectively. Chanrges in vasculature of any vessel and the value given the change included branching of vessels with lymphoid aggregates = 4, vascular thickening = 3, increase in the number of vessels = 2, and dilation = 1. Changes in the bladder tissue and the value given included vacuolization of the urothelium = 5, perivascular infiltrates = 4, increase of collagen, especially in the lamina propria = 3, submucosal edema = 2, serosal changes, especially edema = 1.
Results None of the mice inoculated with B. burgdorferi developed any obvious clinical manifestations during their infection. At autopsy, no gross pathologic changes in the urinary bladder and kidney were evident. Borrelia burgdorferi was isolated from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes (Table 1). The time of isolation varied from only Table 1. Isolation of Borrelia burgdorferifrom the Different Routes ofInoculation
Mouse species RML White-footed mice White-footed mice White-footed mice Grasshopper mice Grasshopper mice HSD nu/nu Total
2 weeks to 8 months postinoculation and resulted from all routes of experimental inoculation including intraperitoneal, subcutaneous, and intranasal. Cultures of bladders from the HSD nu/nu mice were usually positive for spirochetes after only 5 days, whereas cultures of the bladder from the other mice took 1 to 2 weeks before spirochetes were detectable by darkfield microscopy. Spirochetes were not isolated from the bladder of the three grasshopper mice inoculated with the higher-passaged B. burgdorferi (CA-2-87). Surprisingly, we were unable to isolate spirochetes from any of the kidneys from the same 35 mice that had an infected bladder. All attempts to demonstrate spirochetes in paraffinembedded sections of the urinary bladder using immunohistochemical and immunofluorescence stains were unsuccessful, even though the latter technique has worked for us previously.10 Spirochetes were detected, however, by immunofluorescence stains used on imprint smears of the bladder fixed with methanol (Figure 1). The pathologic changes observed most frequently in the bladder of infected mice included the presence of lymphoid aggregates, vascular changes, and perivascular infiltrates (Figures 2-4). Of the 29 mice with infected bladders that were examined histologically after intraperitoneal inoculation, 20 (69%) had lymphoid aggregates, 20 (69%) had perivascular infiltrates, and 20 (69%) had vascular changes; 27 (93%) of the 29 mice had one or more of these alterations, and only 2 (7%) mice had bladders free of such changes. These pathologic changes were not observed in any of 16 control mice that were not infected, including two RML BALB/c mice, two whitefooted mice, two grasshopper mice inoculated with PBS; three grasshopper mice inoculated with noninfectious live spirochetes; 10 nude mice 53 to 75 days old that were not inoculated with anything. There was no obvious correlation with the pathologic changes and duration of infection; however, the HSD nu/nu mice killed after only 14 to 21 days were free of these abnormalities. Of the mice with lymphoid aggregates visible in one section of the bladder, nine (45%) of them had only one aggregate,
Urinary Bladder and Left Kidney of Various Mice Species after
Strain of inoculum
No. of mice
Route of inoculation
Sh-2-82 Sh-2-82 ECM-NY-86 ECM-NY-86 Sh-2-82 CA-2-87 Sh-2-82
6 6 3 3 6 3
i.p. i.p.
11
38
s.c. i.n.
i.p. i.p. i.p.
Duration of infection 8 months 21 days 4 months 4 months 4 months 4 months 14-123 days
No. of isolations/ No. of samples Urinary bladder Kidney 6/6 0/6 6/6 0/6 3/3 0/3 3/3 0/3 6/6 0/6 0/3 0/3 11/11 0/11 35/38 0/38
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Figure 1. Imprint smear of the urinary bladder wall of a white-footed mouse 4 months postinfection. Borrelia burgdorferi stained by direct immunofluorescence antibody and counter stained with Evans blue. Original magnification x 1000.
whereas 11 (55%) mice had two to five aggregates per section. Most of the aggregates were relatively small, constituting less than 25% of 1 HPF. The most common vascular changes included an increase in the number of vessels (18 mice) and thickening of the vessel wall (10 mice). Most of the pathologic changes were seen in the lamina propria. Other types of pathologic changes were observed in some of the mice, including vacuolization of
Figure 2. A lymphoid aggregate in the lamina propria of the urinary bladder of a grasshopper mouse 4 months postinfection. Note the vacuolization in the epithelium. H + E stain. Original magnification x100.
the urothelium, increase in collagen, and submucosal and serosal edema. These abnormalities also were observed in some of the uninfected mice, and it is therefore difficult to ascribe these changes to spirochetal infection. There were no obvious differences observed in the pathologic changes of the bladder among the different species or strains of mice examined. Most individuals in each group of infected mice had sections of the urinary bladder that contained one or more of the abnormalities
'~
Figure 3. Higher magnification of the same lymphoid aggregate in Figure 2 displaying the branching of vessels at the bottom of the aggregate. Original magnification X400.
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Figure 4. Mononuclear cells surrounding a peripheral ganglion in the outer layer of the urinary bladder of a RML mouse 8 months postinfection. Note the extensive edema. H + E stain. Original magnification X250.
described above. All 6 (100%) of the grasshopper mice infected with low-passaged spirochetes, however, had lymphoid aggregates, whereas only six (55%) of the 11 HSD nu/nu infected mice had such aggregates detectable in sections of the bladder. A methyl-green-pyronin stain was used to identify mast and plasma cells associated with the B. burgdorferi-induced cystitis in the mice. Although mast cells were seen in the vicinity of perivascular infiltrates, lymphoid aggregates, and few perineural infiltrations, a positive correlation between the number of mast cells and cystitis was not observed. Variation in the number of mast cells appeared to be associated with the species or strain of mouse rather than being associated with infection with B. burgdorferi. Moreover, there was no obvious increase in the number of mast cells in grasshopper mice inoculated with the infectious low-passage strain compared with those animals inoculated with the noninfectious highpassage strain of the spirochete.
Discussion Infection with Borrelia burgdorferi in the urinary bladder of 29 mice of different strains and species resulted in a detectable cystitis in most (93%) of the animals. Each of the three most frequently observed pathologic lesions, lymphoid aggregates, vascular branching and thickening, and mononuclear perivascular infiltrates, were equally prevalent (69%) and found most often in the lamina propria. Such changes were not observed in any of 16 control mice. The lymphoid aggregates and perivascular infiltrates consisted primarily of lymphocytes, some plasma cells, and rarely mast cells, characteristic of an inflammation process, and similar to the pathologic changes described for infection by Treponema paIlidum.3" This type of cellular and vessel proliferation in the infected urinary
bladders is also suggestive of ongoing immunologic stimulation indicative of a chronic type of infection. The low number of plasma cells and the absence of proliferated (= multinucleated giant or epithelioid cells) and activated macrophages suggest that B. burgdorferi may not be a potent mediator of inflammation. Additionally, the lymphoid aggregates were not granulomatous. Granulomas are due to the activation and accumulation of T cells, a process that stimulates the fusion of monocytes or macrophages through interleukin-4.35 Although nude mice are T cell deficient,36 nearly one-half of the HSD nu/nu mice infected with B. burgdorferi produced lymphoid aggregates about the same size (= size 1) as did the immunocompetent mice. Our findings would support that the lack of T cells does not significantly influence either the clinical course or the pathogenesis of Lyme disease, at least as demonstrated by the types of animals used in this study. In human interstitial cystitis, the histopathology is relatively nonspecific. Round cell infiltrates are observed both diffusely and in aggregates in the bladder lamina submucosa or lamina muscularis accompanied by vasodilation and edema in the lamina submucosa.37 Fibrosis of the submucosal and muscular layer of the bladder wall is a late sequelae of the disease.38 The histopathologic features of cystitis in mice infected with B. burgdorferi were very similar. The most common lesions are one or more lymphoid aggregates, perivascular infiltrates of mononuclear cells, and an increase in the number of vessels. In humans, interstitial cystitis is also clinically defined by sterile urine cultures.39 Negative urine cultures also have been demonstrated in B. burgdorferi-infected P. Ieucopus,1023 Syrian hamsters,11 and Lewis rats.40 The presence of Lyme spirochetes in the urine of wild whitefooted mice22 may have resulted from coinfections with Babesia microti, creating an acute catarrhalic infection. A variety of mechanisms may be involved in the development of spirochetal tissue tropism. Certain receptors could be required to mediate B. burgdorferi host cell attachment like the outer surface layer of mucopolysaccharide material (hyaluronic acid and chondroitin sulfate) in T. pallidum infection.34 Certain tissues have higher concentrations of mucopolysaccharide ground substance like dermis, testis, or the immediate vicinity of blood vessels,34 all of which are involved in B. burgdorferi infection. Recently, one study demonstrated that the tissue invasiveness of B. burgdorferi may be unrelated to hyaluronidase activity.41 Another important factor could be the fibronectin concentration of the various tissues. Fibronectin staining is dense in the vicinity of basement membranes and other areas in the lamina propria of the urinary bladder.42 Binding to this major connective tissue protein seems to play an important role in the pathogenesis of syphilis. Treponema pallidum may use fibronectin
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to attach to host surfaces, or it may become coated with it and avoid the host's immune response.43 The pathogenesis of B. burgdorferi-induced cystitis reflects invasive properties of the spirochete, but the actual method of invasion into the urinary bladder is unknown. Borrelia burgdorferi could gain access to the urinary bladder by the hematogenous route or by urinogenous dissemination through the kidney. Because the bladder wall is in direct contact with urine, the causative agent may come from the urine and penetrate into the deeper layer of the bladder, giving rise to inflammation. Yet the relative integrity of the urothelium as seen by light microscopy, and the often negative kidney cultures, suggest that the infection is not through the urine, but more likely through the blood. Treponema pallidum can penetrate endothelial monolayer barriers by passing through intercellular junctions,44 and there is evidence that B. burgdorferi may do the same.45 Again, the urinary bladder was a consistent source for isolating B. burgdorferi from additional species and strains of mice as was reported previously for whitefooted mice.10'23 Our negative cultures of kidney differed, however, from other results both in mice,2223 and rats,16'40 in which infection of the kidney varied from 18% to 100% at various times after experimental inoculation. Our results document the histopathologic changes associated with cystitis in mice. The possibility of a similar clinical picture in humans with Lyme disease is worthy of future consideration.
Acknowledgments The authors thank Robert Evans and Gary Hettrick for photography, Betty Kester for secretarial help, and Dr. Markus Czub for manuscript review.
References 1. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP: Lyme disease: A tick-borne spirochetosis? Science 1982, 216:1317-1319 2. Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorfer W, Schmidt GP, Johnson E, Malawista SE: The spirochetal etiology of Lyme disease. N Engi J Med 1983, 308:733-740 3. Steere AC: Lyme disease. N Engl J Med 1989,321:586-596 4. Donahue JG, Piesman J, Spielman A: Reservoir competence of white-footed mice for Lyme disease spirochetes. Am J Trop Med Hyg 1987, 36:92-96 5. Levine JF, Wilson ML, Spielman A: Mice as reservoirs of the Lyme disease spirochete. Am J Trop Med Hyg 1985, 34: 355-360 6. Duray PH, Steere AC: The spectrum of organ and systems pathology in human Lyme disease. Zentralbi Bakteriol Mikrobiol Hyg [A] 1986, 263:169-178
7. Duray PH: Histopathology of clinical phases of human Lyme disease. Rheum Dis Clin North Am 1989, 15:691-710 8. Duray PH: Clinical pathologic correlations of Lyme disease. Rev Infect Dis 1989,11:1487-1493 9. Schwan TG, Burgdorfer W, Garon CF: Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect Immun 1988, 56:1831-1836 10. Schwan TG, Burgdorfer W, Schrumpf ME, Karstens RH: The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus). J Clin Microbiol 1988, 26:893-895 11. Johnson RC, Marek N, Kodner C: Infection of Syrian hamsters with Lyme disease spirochetes. J Clin Microbiol 1984, 20:1099-1101 12. Barthold SW: Infectivity of Borrelia burgdorferi relative to route of inoculation and genotype in laboratory mice. J Infect Dis 1991, 163:419-420 13. Barthold SW, Beck DS, Hansen GM, Terwilliger GA, Moody KD: Lyme borreliosis in selected strains and ages of laboratory mice. J Infect Dis 1990, 162:133-138 14. Duray PH, Johnson RC: The histopathology of experimentally infected hansters with the Lyme disease spirochete, Borrelia burgdorferi (42251). Proc Soc Exp Biol Med 1986, 181:263-269 15. Schaible VE, Kramer MD, Museteanu C, Zimmer G, Mossman H, Simon MM: The severe combined immunodeficiency scid mouse: A laboratory model for the analysis of Lyme arthritis and carditis. J Exp Med 1989, 170:1427-1432 16. Barthold SW, Moody KD, Terwilliger GA, Duray PH, Jacoby RO, Steere AC: Experimental Lyme arthritis in rats infected with Borrelia burgdorferi. J Infect Dis 1988, 157:842-846 17. Barthold SW, Moody KD, Terwilliger GA, Jacoby RO, Steere AS: An animal model for Lyme arthritis. Ann NY Acad Sci 1988, 539:264-273 18. Schmitz JL, Schell RF, Hejka A, England DM, Konick L: Induction of Lyme arthritis in LSH hamsters. Infect Immun 1988, 56:2336-2342 19. Johnson RC, Kodner C, Russell M: In vitro and in vivo susceptibility of the Lyme disease spirochete, Borrelia burgdorferi, to four antimicrobial agents. Antimicrob Agents Chemother 1987, 31:164-167 20. Johnson RC, Kodner C, Russell M: Active immunization of hamsters against experimental infection with Borrelia burgdorferi. Infect Immun 1986, 54:897-898 21. Fikrig E, Barthold SW, Kantor FS, Flavell RA: Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 1990, 250:553-556 22. Bosler EM, Schulze TL: The prevalence and significance of Borreia burgdorferi in the urine of feral reservoir hosts. Zentralbl Bakteriol Mikrobiol Hyg [A]. 1986, 263:40-44 23. Callister SM, Agger WA, Schell RF, Brand KM: Efficacy of the urinary bladder for isolation of Borrelia burgdorferi from naturally infected, wild Peromyscus leucopus. J Clin Microbiol 1989, 27:773-774 24. Goodman JL, Jurkovich P, Kodner C, Johnson RC: Persistent cardiac and urinary tract infections with Borrelia burg-
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34. Fitzgerald TJ: Pathogenesis and immunology of Treponema pallidum. Annu Rev Microbiol 1981, 35:29-54 35. McInnes A, Rennick DM: Interleukin 4 induces cultured monocytes/macrophages to form multinucleated cells. J Exp Med 1988,167:598-611 36. Kindred B: Nude mice in immunology. Prog Allergy 1979, 26:137-238 37. Oravisto KJ: Interstitial cystitis as an autoimmune disease. Eur Urol 1980, 6:10-13 38. Holm-Bentzen M, Lose G: Pathology and pathogenesis of interstitial cystitis. Urol Suppl 1987, 39:8-13 39. Sant GR: Interstitial cystitis: Pathophysiology, clinical evaluation, and treatment. Ann Urol 1989, 3:171-196 40. Moody KD, Barthold SW, Terwilliger GA, Beck DS, Hansen GM, Jacoby RO: Experimental chronic Lyme borreliosis in Lewis rats. Am J Trop Med Hyg 1990, 42:165-174 41. Kimsey RB, Spielman A: Motility of Lyme disease spirochetes in fluids as viscous as the extracellular matrix. J Infect Dis 1990,162:1205-1208 42. Gardiner RA, Seymour GJ, Lavin MF, Gemmell E, Hazan G: Immunohistochemical analysis of the human bladder. Br J Urol 1986, 58:19-25 43. Finlay BB, Falkow S: Common themes in microbial pathogenicity. Microbiol Rev 1989, 53:210-230 44. Thomas DD, Navab M, Haake DA, Fogelman AM, Miller JN, Lovett MA. Treponema pallidum invades intercellular junctions of endothelial cell monolayers. Proc Natl Acad Sci USA 1988, 85:3608-3612 45. Thomas DD, Comstock LE: Interaction of Lyme disease spirochetes with cultured eucaryotic cells. Infect Immun 1989, 57:1324-1 326
Cystitis Induced by Infection with the Lyme Disease Spirochete, Borrelia burgdorferi, in Mice Stephanie Czub,* Paul H. Duray,t Rex E. Thomas,* and Tom G. Schwan* From the Department of Health and Human Services,* Public Health Service, National Institutes ofHealth, National Institute ofAllergy and Infectious Diseases, Laboratory of Vectors and Pathogens, Arthropod-borne Diseases Section, Hamilton, Montana, and Fox Chase Cancer Center,t
Philadelphia, Pennsylvania
Previous studies have demonstrated that the urinary bladder is a consistent source for isolating the Lyme disease spirochete, Borrelia burgdorferi,from both experimentally infected and naturally exposed rodents. We examined histopathologic changes in the urinary bladder of different types of rodents experimentally infected with Lyme spirochetes, including BALBIc mice (Mus musculus), nude mice (M. musculus), white-footed mice (Peromyscus leucopus), and grasshopper mice (Onychomys leucogaster). Animals were inoculated intraperitoneally, subcutaneously, or intranasally with low-passaged spirochetes, highpassaged spirochetes,; or phosphate-buffered saline. At various times after inoculation, animals were killed and approximately one-half of each urinary bladder and kidney were cultured separately in BSKII medium while the other half of each organ was prepared for histologic examination. Spirochetes were cultured from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes while we were unable to isolate spirochetes from any kidneys of the same mice. The pathologic changes observed most frequently in the urinary bladder of the infected mice were the presence of lymphoid aggregates, vascular changes, including an increase in the number of vessels and thickening of the vessel walls, and perivascular infiltrates. Our results demonstrate that nearly all individuals (93%) of the four types of mice e-xamined had a cystitis associated with spiro-
chetal infection. (AmjPathol 1992, 141:1173-1179)
Borrelia burgdorferi, the causative agent of Lyme disease, is a tick-borne spirochete infecting humans and a variety of both domestic and wild animals throughout
many regions of the world.1-3 In the hyperendemic regions of the northem midwest and northeastern United States, the white-footed mouse, Peromyscus leucopus, is the primary reservoir for the spirochete and an important host for immature Ixodes dammini ticks that transmit the spirochete.4'5 Lyme disease in humans is a multisystem disorder that varies tremendously in its clinical presenta-
tion.2'S
Numerous reports have demonstrated the usefulness of various rodents as experimental animals for investigating the infectiousness of B. burgdorferi,9-13 to examine specific aspects of the pathogenesis of Lyme disease,1618 to determine in vivo antibiotic susceptibility,19 and to examine potential vaccines. 2021 Studies with animals and reports of urine of humans suggest that either the urinary bladder or the urinary tract may be infected with the Lyme spirochete. Previous investigations have shown that B. burgdorferi may be present in the urine of naturally infected white-footed mice,22 and that the urinary bladder of experimentally infected and naturally infected rodents often produces spirochetes when this organ is triturated and inoculated into BSK-11 medium.10'23'24 Antigens of B. burgdorferi have been detected in the urine of both laboratory mice experimentally infected with B. burgdorferi and humans with Lyme disease,25, "6and B. burgdorferi has been demonstrated in the bladder wall of white-footed mice by direct immunofluorescence staining.10 As well, the amplification of B. burgdorferi DNA using the polymerase chain reaction has been used recently to detect spirochetes in the urine of humans with Lyme disease,27 and bladder function disturbance leading to urine retention has also been reported.2e The frequent infection of spirochetes in the uriDr. Czub's current address is the Institute of Pathology, University of
Wurzburg, Germany. Dr. Duray's current address is the Department of Pathology, Brgham and Women's Hospital, Harvard Medical School, Boston, Massachusetts. Dr. Thomas' current address is Paravax, Inc., Fort Collins, Colorado. Accepted for publication May 14, 1992. Address reprint requests to Dr. Tom G. Schwan, Department of Health and Human Services, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Vectors and Pathogens, Arthropod-bome Diseases Section, Hamilton, MT 59840.
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nary bladder of experimental and wild rodents has raised the question as to whether some cases of interstitial cystitis in humans might be caused by B. burgdorferi.'9 Because of the previous studies cited above, demonstrating that the urinary bladder of rodents often produces isolations of B. burgdorferi, including mice that have been infected for 16 months,30 the objective of the current study was to determine if infection by Lyme spirochetes resulted in any histopathologic changes in this organ.
Materials and Methods
Animals and Housing Conditions The following strains and species of mice were infected with B. burgdorferi: laboratory mice (Mus musculus, RML BALB/c/Ann), grasshopper mice (Onychomys Ieucogaster), white-footed mice, and nude Harlan SpragueDawley mice (HSD nu/nu, M. musculus). All animals were obtained from the Rocky Mountain Laboratories animal production facility. Both sexes were used, and all animals were 1 to 3 months old when inoculated. RML and HSD nu/nu mice were kept in groups of four to five animals per cage. The grasshopper mice and white-footed mice were kept separately with only one animal per cage.
Strains of B. burgdorferi and Animal Inoculation Borrelia burgdorferi strains Sh-2-82, CA-2-87, and ECMNY-86 were described elsewhere.10 Briefly, Sh-2-82 originated from Ixodes dammini ticks from Shelter Island, New York; CA-2-87 originated from pacificus ticks from Tulare County, California; ECM-NY-86 originated from a skin biopsy from a EM lesion of a human patient in Southampton, New York. Fresh, low-passaged cultures (passage 5) of Sh-2-82 and ECM-NY-86 were prepared in BSK-11 medium,31 removed from the medium by centrifugation, and resuspended in PBS-5 mmol/l MgC12 (pH 7.4), and counted in a Petroff-Hauser bacteria counting chamber. A higher-passaged (passage 15) noninfectious culture of CA-2-87 was prepared in the same manner. Animals infected with the low-passaged strain were inoculated by one of three routes including intraperitoneal, subcutaneous, or intranasal inoculation with approximately 7 x 107 B. burgdorferi in PBS-5 mmol/l MgC12 (pH 7.4). Animals inoculated with the high-passaged strain were injected intraperitoneally with the same number of spirochetes and caged separately from mice inoculated with low-passaged spirochetes. Six mice (two of each species) were also inoculated with PBS intraperitoneally for negative controls and kept in separate cages in
the same room. The animals were examined twice per week for their health status. Between 14 days and 8 months postinfection, the mice were anesthetized with ether, killed, and the kidney and urinary bladder were removed under sterile conditions. Ten uninoculated nude mice were also examined.
Cultivation of Borrelia burgdorferi One half of each kidney and urinary bladder was used for the histologic examination (see below), and the other half was used to isolate spirochetes as described previously.10 Briefly, each tissue was triturated in 1 ml BSK-11 medium in a glass tissue grinder. Half of this suspension was inoculated into a tube containing 9 ml plain BSK-ll medium, and the other half into a tube containing 9 ml BSK-1l medium plus phosphomycin (100 ,ug/ml) and rifampin (50 ,ug/ml). The cultures were incubated at 340C and checked by darkfield microscopy every few days for 2 to 3 weeks. Organs of control animals were treated in the same way. Bladders from the 10 uninoculated nude mice were only examined histologically.
Tissue Processing Imprint smears of one half of the urinary bladder and the left kidney from infected and normal animals were fixed with methanol and stained by a direct immunofluorescence assay using rabbit anti-B. burgdorferi antibodies labeled with fluorescein isothiocyanate (FITC). Afterwards, the organs were prepared for sectioning using standard methods, cut in 4-,. serial sections and mounted on slides, and examined by light microscopy.
Staining Procedures Hematoxylin and eosin (H&E) stain was used on all slides. Methyl-green-pyronin stain (Sigma) was used to detect plasma cells and mast cells. Immunohistochemical stains (APAAP, ABC peroxidase), direct immunofluorescence with rabbit anti-B. burgdorferi antibody labeled with FITC, and indirect immunofluorescence with monoclonal anti-B. burgdorferi outer surface protein A antibody H5332,32 monoclonal anti-flagellin antibody H9724,33 and polyclonal rabbit anti-B. burgdorferi antibody were used to detect B. burgdorferi in the tissues.
Analysis of Histologic Changes in the Urinary Bladder Sections from the urinary bladder of 48 mice were examined for histologic abnormalities, including 29 from mice
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inoculated intraperitoneally with infectious spirochetes, three from mice inoculated intraperitoneally with noninfectious spirochetes, six from mice inoculated with only PBS, and 10 uninoculated nude mice. Part of the same bladder from all the mice (excluding the 10 nude control mice) were also cultured in BSK-ll. The identity of the mice and the results of cultivation were not known when the slides were examined and the histologic changes quantified. Pathologic changes in the bladder included the number and size of lymphoid aggregates, changes in vasculature, and alterations in bladder tissues. Tissues were scored to quantify the various changes and included the following: the number of lymphoid aggregates per section varied from 0, 1, 2, 3, 4, or more and received scores of 0 to 5, respectively. Sizes of the aggregates measured with an ocular micrometer for relative size to one highpower field (HPF, 400x magnification), varied from less than 25%, 25% to 50%, 50% to 75%, 75% to 100%, and greater than 100% of HPF and received scores of 1 to 5, respectively. Chanrges in vasculature of any vessel and the value given the change included branching of vessels with lymphoid aggregates = 4, vascular thickening = 3, increase in the number of vessels = 2, and dilation = 1. Changes in the bladder tissue and the value given included vacuolization of the urothelium = 5, perivascular infiltrates = 4, increase of collagen, especially in the lamina propria = 3, submucosal edema = 2, serosal changes, especially edema = 1.
Results None of the mice inoculated with B. burgdorferi developed any obvious clinical manifestations during their infection. At autopsy, no gross pathologic changes in the urinary bladder and kidney were evident. Borrelia burgdorferi was isolated from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes (Table 1). The time of isolation varied from only Table 1. Isolation of Borrelia burgdorferifrom the Different Routes ofInoculation
Mouse species RML White-footed mice White-footed mice White-footed mice Grasshopper mice Grasshopper mice HSD nu/nu Total
2 weeks to 8 months postinoculation and resulted from all routes of experimental inoculation including intraperitoneal, subcutaneous, and intranasal. Cultures of bladders from the HSD nu/nu mice were usually positive for spirochetes after only 5 days, whereas cultures of the bladder from the other mice took 1 to 2 weeks before spirochetes were detectable by darkfield microscopy. Spirochetes were not isolated from the bladder of the three grasshopper mice inoculated with the higher-passaged B. burgdorferi (CA-2-87). Surprisingly, we were unable to isolate spirochetes from any of the kidneys from the same 35 mice that had an infected bladder. All attempts to demonstrate spirochetes in paraffinembedded sections of the urinary bladder using immunohistochemical and immunofluorescence stains were unsuccessful, even though the latter technique has worked for us previously.10 Spirochetes were detected, however, by immunofluorescence stains used on imprint smears of the bladder fixed with methanol (Figure 1). The pathologic changes observed most frequently in the bladder of infected mice included the presence of lymphoid aggregates, vascular changes, and perivascular infiltrates (Figures 2-4). Of the 29 mice with infected bladders that were examined histologically after intraperitoneal inoculation, 20 (69%) had lymphoid aggregates, 20 (69%) had perivascular infiltrates, and 20 (69%) had vascular changes; 27 (93%) of the 29 mice had one or more of these alterations, and only 2 (7%) mice had bladders free of such changes. These pathologic changes were not observed in any of 16 control mice that were not infected, including two RML BALB/c mice, two whitefooted mice, two grasshopper mice inoculated with PBS; three grasshopper mice inoculated with noninfectious live spirochetes; 10 nude mice 53 to 75 days old that were not inoculated with anything. There was no obvious correlation with the pathologic changes and duration of infection; however, the HSD nu/nu mice killed after only 14 to 21 days were free of these abnormalities. Of the mice with lymphoid aggregates visible in one section of the bladder, nine (45%) of them had only one aggregate,
Urinary Bladder and Left Kidney of Various Mice Species after
Strain of inoculum
No. of mice
Route of inoculation
Sh-2-82 Sh-2-82 ECM-NY-86 ECM-NY-86 Sh-2-82 CA-2-87 Sh-2-82
6 6 3 3 6 3
i.p. i.p.
11
38
s.c. i.n.
i.p. i.p. i.p.
Duration of infection 8 months 21 days 4 months 4 months 4 months 4 months 14-123 days
No. of isolations/ No. of samples Urinary bladder Kidney 6/6 0/6 6/6 0/6 3/3 0/3 3/3 0/3 6/6 0/6 0/3 0/3 11/11 0/11 35/38 0/38
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Figure 1. Imprint smear of the urinary bladder wall of a white-footed mouse 4 months postinfection. Borrelia burgdorferi stained by direct immunofluorescence antibody and counter stained with Evans blue. Original magnification x 1000.
whereas 11 (55%) mice had two to five aggregates per section. Most of the aggregates were relatively small, constituting less than 25% of 1 HPF. The most common vascular changes included an increase in the number of vessels (18 mice) and thickening of the vessel wall (10 mice). Most of the pathologic changes were seen in the lamina propria. Other types of pathologic changes were observed in some of the mice, including vacuolization of
Figure 2. A lymphoid aggregate in the lamina propria of the urinary bladder of a grasshopper mouse 4 months postinfection. Note the vacuolization in the epithelium. H + E stain. Original magnification x100.
the urothelium, increase in collagen, and submucosal and serosal edema. These abnormalities also were observed in some of the uninfected mice, and it is therefore difficult to ascribe these changes to spirochetal infection. There were no obvious differences observed in the pathologic changes of the bladder among the different species or strains of mice examined. Most individuals in each group of infected mice had sections of the urinary bladder that contained one or more of the abnormalities
'~
Figure 3. Higher magnification of the same lymphoid aggregate in Figure 2 displaying the branching of vessels at the bottom of the aggregate. Original magnification X400.
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Figure 4. Mononuclear cells surrounding a peripheral ganglion in the outer layer of the urinary bladder of a RML mouse 8 months postinfection. Note the extensive edema. H + E stain. Original magnification X250.
described above. All 6 (100%) of the grasshopper mice infected with low-passaged spirochetes, however, had lymphoid aggregates, whereas only six (55%) of the 11 HSD nu/nu infected mice had such aggregates detectable in sections of the bladder. A methyl-green-pyronin stain was used to identify mast and plasma cells associated with the B. burgdorferi-induced cystitis in the mice. Although mast cells were seen in the vicinity of perivascular infiltrates, lymphoid aggregates, and few perineural infiltrations, a positive correlation between the number of mast cells and cystitis was not observed. Variation in the number of mast cells appeared to be associated with the species or strain of mouse rather than being associated with infection with B. burgdorferi. Moreover, there was no obvious increase in the number of mast cells in grasshopper mice inoculated with the infectious low-passage strain compared with those animals inoculated with the noninfectious highpassage strain of the spirochete.
Discussion Infection with Borrelia burgdorferi in the urinary bladder of 29 mice of different strains and species resulted in a detectable cystitis in most (93%) of the animals. Each of the three most frequently observed pathologic lesions, lymphoid aggregates, vascular branching and thickening, and mononuclear perivascular infiltrates, were equally prevalent (69%) and found most often in the lamina propria. Such changes were not observed in any of 16 control mice. The lymphoid aggregates and perivascular infiltrates consisted primarily of lymphocytes, some plasma cells, and rarely mast cells, characteristic of an inflammation process, and similar to the pathologic changes described for infection by Treponema paIlidum.3" This type of cellular and vessel proliferation in the infected urinary
bladders is also suggestive of ongoing immunologic stimulation indicative of a chronic type of infection. The low number of plasma cells and the absence of proliferated (= multinucleated giant or epithelioid cells) and activated macrophages suggest that B. burgdorferi may not be a potent mediator of inflammation. Additionally, the lymphoid aggregates were not granulomatous. Granulomas are due to the activation and accumulation of T cells, a process that stimulates the fusion of monocytes or macrophages through interleukin-4.35 Although nude mice are T cell deficient,36 nearly one-half of the HSD nu/nu mice infected with B. burgdorferi produced lymphoid aggregates about the same size (= size 1) as did the immunocompetent mice. Our findings would support that the lack of T cells does not significantly influence either the clinical course or the pathogenesis of Lyme disease, at least as demonstrated by the types of animals used in this study. In human interstitial cystitis, the histopathology is relatively nonspecific. Round cell infiltrates are observed both diffusely and in aggregates in the bladder lamina submucosa or lamina muscularis accompanied by vasodilation and edema in the lamina submucosa.37 Fibrosis of the submucosal and muscular layer of the bladder wall is a late sequelae of the disease.38 The histopathologic features of cystitis in mice infected with B. burgdorferi were very similar. The most common lesions are one or more lymphoid aggregates, perivascular infiltrates of mononuclear cells, and an increase in the number of vessels. In humans, interstitial cystitis is also clinically defined by sterile urine cultures.39 Negative urine cultures also have been demonstrated in B. burgdorferi-infected P. Ieucopus,1023 Syrian hamsters,11 and Lewis rats.40 The presence of Lyme spirochetes in the urine of wild whitefooted mice22 may have resulted from coinfections with Babesia microti, creating an acute catarrhalic infection. A variety of mechanisms may be involved in the development of spirochetal tissue tropism. Certain receptors could be required to mediate B. burgdorferi host cell attachment like the outer surface layer of mucopolysaccharide material (hyaluronic acid and chondroitin sulfate) in T. pallidum infection.34 Certain tissues have higher concentrations of mucopolysaccharide ground substance like dermis, testis, or the immediate vicinity of blood vessels,34 all of which are involved in B. burgdorferi infection. Recently, one study demonstrated that the tissue invasiveness of B. burgdorferi may be unrelated to hyaluronidase activity.41 Another important factor could be the fibronectin concentration of the various tissues. Fibronectin staining is dense in the vicinity of basement membranes and other areas in the lamina propria of the urinary bladder.42 Binding to this major connective tissue protein seems to play an important role in the pathogenesis of syphilis. Treponema pallidum may use fibronectin
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to attach to host surfaces, or it may become coated with it and avoid the host's immune response.43 The pathogenesis of B. burgdorferi-induced cystitis reflects invasive properties of the spirochete, but the actual method of invasion into the urinary bladder is unknown. Borrelia burgdorferi could gain access to the urinary bladder by the hematogenous route or by urinogenous dissemination through the kidney. Because the bladder wall is in direct contact with urine, the causative agent may come from the urine and penetrate into the deeper layer of the bladder, giving rise to inflammation. Yet the relative integrity of the urothelium as seen by light microscopy, and the often negative kidney cultures, suggest that the infection is not through the urine, but more likely through the blood. Treponema pallidum can penetrate endothelial monolayer barriers by passing through intercellular junctions,44 and there is evidence that B. burgdorferi may do the same.45 Again, the urinary bladder was a consistent source for isolating B. burgdorferi from additional species and strains of mice as was reported previously for whitefooted mice.10'23 Our negative cultures of kidney differed, however, from other results both in mice,2223 and rats,16'40 in which infection of the kidney varied from 18% to 100% at various times after experimental inoculation. Our results document the histopathologic changes associated with cystitis in mice. The possibility of a similar clinical picture in humans with Lyme disease is worthy of future consideration.
Acknowledgments The authors thank Robert Evans and Gary Hettrick for photography, Betty Kester for secretarial help, and Dr. Markus Czub for manuscript review.
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