Vol. 30, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 3016-3018 0095-1137/92/113016-03$02.00/0 Copyright © 1992, American Society for Microbiology
Experimental Borrelia burgdorferi Infection of Outbred Mice TOSHIYUKI MASUZAWA,1,2* YOSHIHITO BEPPU,' HIROKI KAWABATA,1 YASUTAKE YANAGIHARA,1 YOSHIHISA IWAMOTO,' TADAYORI SHIMIZU,' AND RUSSELL C. JOHNSON2
Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan, 1 and Department of Microbiology, Medical School, University of Minnesota, 420 Delaware Street S.E., Minneapolis, Minnesota 554552 Received 14 May 1992/Accepted 14 August 1992
Infectivity of Borrelia burgdorfieri strain 297 in normal outbred ddY mice was examined. Strain 297 was inoculated intraperitoneally in 3-week-old outbred ddY mice. B. burgdorferi was routinely cultured from the heart and urinary bladder 5 to 84 days postinoculation. The combined isolation rate from both heart and urinary bladder was significantly higher than the rate from spleen, kidney, liver, urine, and blood samples. Three- and 10-week-old mice were infected with inocula of 104 and iOs or more, respectively. Passive transfer of undiluted and 10-fold-diluted anti-297 rabbit serum and active immunization of 50 to 100 pg of lyophilized whole cells completely protected mice from infection with B. burgdorferi. These results suggest that the outbred mouse is a convenient model for experimental infection with B. burgdorferi. Several laboratory animal species have been experimentally infected with Borrelia burgdorferi. These include dogs (11), rabbits (7, 15, 16), guinea pigs (16), hamsters (9, 10, 12-14, 23-25), rats (4, 5, 19), inbred mice (2, 3, 6), severe combined immunodeficiency (SCID) mice (20-22), and white-footed mice (Peromyscus leucopus) (18, 26), the primary reservoir of B. burgdorferi in North America. Experimentally infected rats (5), inbred mice (3), SCID mice (22), and irradiated hamsters (24) are useful for pathological studies because they frequently display arthritis and/or carditis. Rabbits and guinea pigs infected with B. burgdorferi develop skin lesions resembling human erythema migrans (7, 15, 16). We believe that experimental mouse models are desirable because the feeding and handling of mice are easier than those of other animals. Barthold et al. (3), using various strains of inbred mice, demonstrated that C3HIHeJ, C3H/ HeN, and SWR mice developed polyarthritis after intraperitoneal (i.p.) inoculation with B. burgdorferi N40. Furthermore, in preliminary experiments, they noted that infected infant outbred CD-1 mice displayed a high frequency of arthritis. Schaible et al. (22) demonstrated that B. burgdorferi-infected SCID mice developed polyarthritis and carditis. For the isolation of B. burgdorfen from infected animals, the
spleen, kidney, and blood are routinely cultured. Recently it was demonstrated that the urinary bladder and heart are effective sources for isolating B. burgdorferi from experimentally infected white-footed mice and hamsters (10, 26). To our knowledge, there are no reports of an experimental model using outbred mice. A major advantage of outbred mice is their cheaper price. The present study was designed to establish an experimental model with outbred ddY mice and to evaluate the usefulness of this model by passive and active immunization experiments. Three- and 10-week-old outbred ddY mice and 4-week-old golden hamsters were purchased from SLC Japan (Hamamatsu, Japan). Animals were housed at five per cage at an ambient temperature of 22°C. The test organism used was strain 297, originally isolated from human spinal fluid (27). The virulent strain, passaged seven times in vitro, has been maintained by passage in hamster and cultured twice in BSKII medium (1) at 34°C. Mice and hamsters were inoculated i.p. with 108 cells of B. burgdorfien. Numbers of spirochetes in cultures were determined by the counting method of Stoenner (28). This challenge dose represents approximately 10,000 100% infectious doses of this spirochete. At 14 days after inoculation, mice were sacrificed, and
TABLE 1. Isolation of B. burgdorferi from experimentally infected ddY mice No. of mice with positive sample/no. of mice testedb
Day" Day5 7 14 28 56 84
~
Hat Heart
4/5 5/5 5/5
4/5 5/5 5/5 3/5 5/5 4/4
5/5 3/5
4/5
Total (% positive)
26/30 (87)
a Days postinoculation. b Mice were injected i.p. with B. *
lddr Bladder
26/29 (90)
Heart or
Hbladder 5/5
5/5 5/5 5/5
Spleen
Kidney
4/5
4/5 3/5
1/5
2/5
0/5
0/5
0/5
1/5 0/5 0/5 0/5 0/5
3/30 (10)
1/30 (3)
0/5
1/5 14/30 (47)
30/30 (100)
Urine
1/5 1/5 0/5
5/5 2/5 1/5 1/5
5/5 5/5
Liver
10/30 (33)
1/5
0/5
Blood
2/5 2/5
0/5
0/5
0/5
1/5 5/30 (7)
burgdorfen (108 cells per mouse) on day zero. After 14 days, mice were sacrificed and tissues were cultured in BSKII medium.
Corresponding author. 3016
VOL. 30, 1992
NOTES
TABLE 2. Susceptibility of outbred ddY mice and hamsters to B. burgdorferi infection No. of cells inoculated
101 102
103 104 105 106 107
108
No. positive/no. of animals used (% positive)' 10-wk-old mice 4-wk-old hamsters
3-wk-old mice 1/5 1/5 4/5 5/5 5/5 5/5 5/5
(20) (20) (80) (100) (100) (100) (100)
5/5 (100)
NT' 0/5 (0) 3/5 (60) 4/5 (80) 5/5 (100) 5/5 (100) 5/5 (100)
5/5 (100)
0/5 (0) 2/5 (40) 0/5 (0) 5/5 (100) 4/5 (80) 5/5 (100) 5/5 (100)
5/5 (100)
"Mice and hamsters were challenged i.p. with B. burgdorferi on day zero. After 14 days, animals were sacrificed and heart and urinary bladder were cultured in BSKII medium. b NT, not tested.
blood, urine, hearts, bladders, kidneys, livers, and spleens were cultured in BSKII medium for 4 weeks by the method of Schwan et al. (26). Efficiencies of cultivation of B. burgdorferi 297 from the heart (87%) and the bladder (90%) were significantly higher than that from spleen, kidney, liver, urine, and blood through 5 to 84 days after inoculation of strain 297 (Table 1). Furthermore, isolation efficiency from the heart and the urinary bladder combined was 100% at all dissection days. To determine susceptibility of ddY mice to infection with B. burgdorferi, 3- and 10-week-old ddY mice and 4-week-old hamsters were challenged i.p. At 14 days postinoculation, animals were sacrificed and the heart and the urinary bladder were cultured in BSKII medium. B. burgdorfen was cultured from the heart and/or the bladder of all 3- and 10-week-old mice inoculated with 104 cells, 105 cells, or greater than 105 cells (Table 2). Hamsters inoculated with 104 cells or more were also infected. These results suggest that the sensitivity of outbred ddY mice to infection with B. burgdorferi was much the same as that of hamsters. In the passive immunization experiment, 100 ,ul of undiluted or diluted anti-297 immune rabbit serum or normal rabbit serum was inoculated i.p. into 3-week-old mice on day zero. Preparations of immune rabbit serum were described previously (17). After 20 h, the mice were challenged i.p. with 108 B. burgdorferi cells. Outbred ddY mice receiving passively transferred undiluted and 10-fold-diluted immune serum were completely protected from B. burgdorfen infection (Table 3). On the other hand, immune serum diluted 100-fold or greater and normal rabbit serum could not TABLE 3. Passive immunization of ddY mice with immune rabbit serum" Serum and dilution
Anti-297 Undiluted 1:10 1:100 1:1,000 1:10,000 1:100,000 Normal, undiluted
of mice used
% Protection
0/5 0/5
100 100
5/5 5/5 5/5 5/5 5/5
0 0 0 0 0
a Mice were inoculated i.p. with immune or normal rabbit serum. After 20 h, mice were challenged i.p. with B. bwgdorferi (108 cells per mouse). After 14 days, the heart and urinary bladder were removed from each mouse and cultured in BSKII medium.
3017
TABLE 4. Active immunization of ddY mice with lyophilized
whole cells of B. Dose of vaccine
(AIg [dry wt]/mouse) 10 25 50 100 0 (saline)
burgdorferfi"
No. positive/no. of mice used (% protection) 14 days
28 days
1/5 (80) 1/5 (80) 0/5 (100) 0/5 (100) 5/5 (0)
1/5 (80) 1/5 (80) 0/5 (100) 0/5 (100) 4/4 (0)
aMice were immunized i.p. with lyophilized whole cells of B. burgdorfen, and 14 or 28 days postimmunization, mice were challenged i.p. with B. burgdorferi (108 cells per mouse). After 14 days, heart and urinary bladder tissues were removed from each mouse and cultured in BSKII medium.
prevent infection. In active immunization experiments, lyophilized B. burgdorferi cells were resuspended in saline at concentrations of 100, 250, 500, and 1,000 ,ug/ml. Threeweek-old mice were given a single dose (100 ,ul per mouse) of the vaccine i.p. The mice were challenged i.p. with 108 B. burgdorferi cells at 14 and 28 days after vaccination. On day 14 after challenge, mice were sacrificed 4nd the heart and the urinary bladder were removed and cultured in BSKII medium. i.p. of 10 and 25 ,ug of vaccine protected four of five mice from challenge with 108 cells of B. burgdorferi. Increasing the amount of vaccine to 50 or 100 ,ug elicited a 100% protective response in mice at both postvaccination times. All nine control mice receiving saline injections were culture positive (Table 4). In this study, we evaluated the usefulness of outbred mice as an experimental model for B. burgdorferi infection. We demonstrated that the heart and the urinary bladder of infected mice were excellent sources of B. burgdorferi. On the other hand, isolation from the liver, the blood, and the urine was less productive. This observation is in accord with the results reported previously (10, 26). Especially worthy of notice is that B. burgdorferi was isolated from the heart and/or the urinary bladder of all infected mice. Since this result suggested that cultivation of the heart and the bladder was sufficient to estimate infection in this model, cultivation was restricted to the two organs in the following experiments. Histopathological changes related to Lyme disease were not displayed by the ddY mice except for slight lymphocytic infiltration in the heart (data not shown). Minor histological changes were reported in hamsters (8-10), normal C.B.-17 mice (22), and the hispid cotton rat (8). We believe that these observations are related to the rodents being primarily reservoir hosts for this spirochete in nature. The spirochete was cultured in urine sample from 1 of 30 mice tested. To our knowledge, this is the first report of isolating B. burgdorferi from urine of experimentally infected mice. Sensitivity of mice to infection with strain 297 was much the same as that for hamsters. Their 100% infectious doses were 104 to 105 cells per animal by i.p. inoculation. The sensitivity to infection of the outbred ddY mice was very similar to that of hamnsters and rats reported by other researchers (13, 19). Furthermore, in the passive and active immunization experiments, a dose-dependent protective effect in mice was exhibited. In conclusion, these results indicate that the outbred ddY mice model is very useful and effective for evaluating the effectiveness of vaccination.
3018
NOTES
This study was supported in part by a Grant-in-Aid for Encouragement of Young Scientists no. 02857348 and a Grant-in-Aid for Scientific Research no. 03807025 from the Ministry of Education, Science, and Culture of Japan and Public Health Service grant AI 29739. REFERENCES 1. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. 2. Barthold, S. W. 1991. Infectivity of Borrelia burgdorfeni relative to route of inoculation and genotype in laboratory mice. J. Infect. Dis. 163:419-420. 3. Barthold, S. W., D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody. 1990. Lyme borreliosis in selected strains and ages of laboratory mice. J. Infect. Dis. 162:133-138. 4. Barthold, S. W., K. D. Moody, and D. S. Beck. 1990. Susceptibility of laboratory rats to isolates of Borrelia burgdorferi from different geographic areas. Am. J. Trop. Med. Hyg. 42:596-600. 5. Barthold, S. W., K. D. Moody, G. A. Terwilliger, P. H. Duray, R. 0. Jacoby, and A. C. Steere. 1988. Experimental Lyme arthritis in rats infected with Borrelia burgdorferi. J. Infect. Dis. 157:842-846. 6. Barthold, S. W., D. H. Persing, A. L. Armstrong, and R. A. Peeples. 1991. Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139:263-273. 7. Burgdorfer, W. 1984. The New Zealand White rabbit: an experimental host for infecting ticks with Lyme disease spirochetes. Yale J. Biol. Med. 57:609-612. 8. Burgdorfer, W., and K. L. Gage. 1987. Susceptibility of the hispid cotton rat (Sigmodon hispidus) to the Lyme disease spirochete (Borrelia burgdorferi). Am. J. Trop. Med. Hyg. 37:624-628. 9. Duray, P. H., and R. C. Johnson. 1986. The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorfen (42251). Proc. Soc. Exp. Biol. Med. 181:263-269. 10. Goodman, J. L., P. Jurkovich, C. Kodner, and R. C. Johnson. 1991. Persistent cardiac and urinary tract infections with Borrelia burgdorfen in experimentally infected Syrian hamsters. J. Clin. Microbiol. 29:894-896. 11. Greene, R. T., R. L. Walker, W. L. Nicholson, H. W. Heidner, J. F. Levine, E. C. Burgess, M. Wyand, E. B. Breitschwerdt, and H. A. Berkhoff. 1988. Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorfen) in experimentally and naturally exposed dogs. J. Clin. Microbiol. 26:648-653. 12. Johnson, R. C., C. Kodner, and M. Russell. 1986. Active immunization of hamsters against experimental infection with Borrelia burgdorferi. Infect. Immun. 54:897-898. 13. Johnson, R. C., C. Kodner, and M. Russell. 1986. Passive immunization of hamsters against experimental infection with the Lyme disease spirochete. Infect. Immun. 53:713-714. 14. Johnson, R. C., N. Marek, and C. Kodner. 1984. Infection of Syrian hamsters with Lyme disease spirochetes. J. Clin. Micro-
J. CLIN. MICROBIOL.
biol. 20:1099-1101. 15. Kornblatt, A. N., A. C. Steere, and D. G. Brownstein. 1984. Infection of rabbits with the Lyme disease spirochete. Yale J. Biol. Med. 57:613-614. 16. Krinsky, W. L., S. J. Brown, and P. W. Askenase. 1982. Ixodes dammini: induced skin lesions in guinea pigs and rabbits compared to erythema chronicum migrans in patients with Lyme arthritis. Exp. Parasitol. 53:381-395. 17. Masuzawa, T., Y. Okada, Y. Yanagihara, and N. Sato. 1991. Antigenic properties of Borrelia burgdorferi isolated from bcodes oratus and Ixodespersulcatus in Hokkaido, Japan. J. Clin. Microbiol. 29:1568-1573. 18. Mather, T. N., S. R. Telford III, and G. H. Adler. 1991. Absence of transplacental transmission of Lyme disease spirochetes from reservoir mice (Peromyscus leucopus) to their offspring. J. Infect. Dis. 164:564-567. 19. Moody, K. D., and S. W. Barthold. 1991. Relative infectivity of Borrelia burgdorferi in Lewis rats by various routes of infection. Am. J. Trop. Med. Hyg. 44:135-139. 20. Schaible, U. E., M. D. Kramer, K. Eichmann, M. Modolell, C. Museteanu, and M. M. Simon. 1990. Monoclonal antibodies specific for the outer surface protein A (Osp A) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc. Natl. Acad. Sci. USA 87: 3768-3772. 21. Schaible, U. E., M. D. Kramer, C. W. E. Justus, C. Museteanu, and M. M. Simon. 1989. Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi. Infect. Immun. 57:41-47. 22. Schaible, U. E., M. D. Kramer, C. Museteanu, G. Zimmer, H. Mossmann, and M. M. Simon. 1989. The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis. J. Exp. Med. 170:14271432. 23. Schmitz, J. L., R. F. Schell, A. G. Hejka, and D. M. England. 1990. Passive immunization prevents induction of Lyme arthritis in LSH hamsters. Infect. Immun. 58:144-148. 24. Schmitz, J. L., R. F. Schell, A. G. Hejka, D. M. England, and L. Konick. 1988. Induction of Lyme arthritis in LSH hamsters. Infect. Immun. 56:2336-2342. 25. Schmitz, J. L., R. F. Schell, S. D. Lovrich, S. M. Callister, and J. E. Coe. 1991. Characterization of the protective antibody response to Borrelia burgdorferi in experimentally infected LSH hamsters. Infect. Immun. 59:1916-1921. 26. Schwan, T. G., W. Burgdorfer, M. E. Schrumpf, and R. H. Karsten. 1988. The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally infected white-footed mice (Peromyscus leucopus). J. Clin. Microbiol. 26:893-895. 27. Steere, A. C., M. S. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista. 1983. The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308:733-740. 28. Stoenner, H. G. 1974. Biology of Borrelia hernsii in Kelly medium. Appl. Microbiol. 28:540-543.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 3016-3018 0095-1137/92/113016-03$02.00/0 Copyright © 1992, American Society for Microbiology
Experimental Borrelia burgdorferi Infection of Outbred Mice TOSHIYUKI MASUZAWA,1,2* YOSHIHITO BEPPU,' HIROKI KAWABATA,1 YASUTAKE YANAGIHARA,1 YOSHIHISA IWAMOTO,' TADAYORI SHIMIZU,' AND RUSSELL C. JOHNSON2
Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan, 1 and Department of Microbiology, Medical School, University of Minnesota, 420 Delaware Street S.E., Minneapolis, Minnesota 554552 Received 14 May 1992/Accepted 14 August 1992
Infectivity of Borrelia burgdorfieri strain 297 in normal outbred ddY mice was examined. Strain 297 was inoculated intraperitoneally in 3-week-old outbred ddY mice. B. burgdorferi was routinely cultured from the heart and urinary bladder 5 to 84 days postinoculation. The combined isolation rate from both heart and urinary bladder was significantly higher than the rate from spleen, kidney, liver, urine, and blood samples. Three- and 10-week-old mice were infected with inocula of 104 and iOs or more, respectively. Passive transfer of undiluted and 10-fold-diluted anti-297 rabbit serum and active immunization of 50 to 100 pg of lyophilized whole cells completely protected mice from infection with B. burgdorferi. These results suggest that the outbred mouse is a convenient model for experimental infection with B. burgdorferi. Several laboratory animal species have been experimentally infected with Borrelia burgdorferi. These include dogs (11), rabbits (7, 15, 16), guinea pigs (16), hamsters (9, 10, 12-14, 23-25), rats (4, 5, 19), inbred mice (2, 3, 6), severe combined immunodeficiency (SCID) mice (20-22), and white-footed mice (Peromyscus leucopus) (18, 26), the primary reservoir of B. burgdorferi in North America. Experimentally infected rats (5), inbred mice (3), SCID mice (22), and irradiated hamsters (24) are useful for pathological studies because they frequently display arthritis and/or carditis. Rabbits and guinea pigs infected with B. burgdorferi develop skin lesions resembling human erythema migrans (7, 15, 16). We believe that experimental mouse models are desirable because the feeding and handling of mice are easier than those of other animals. Barthold et al. (3), using various strains of inbred mice, demonstrated that C3HIHeJ, C3H/ HeN, and SWR mice developed polyarthritis after intraperitoneal (i.p.) inoculation with B. burgdorferi N40. Furthermore, in preliminary experiments, they noted that infected infant outbred CD-1 mice displayed a high frequency of arthritis. Schaible et al. (22) demonstrated that B. burgdorferi-infected SCID mice developed polyarthritis and carditis. For the isolation of B. burgdorfen from infected animals, the
spleen, kidney, and blood are routinely cultured. Recently it was demonstrated that the urinary bladder and heart are effective sources for isolating B. burgdorferi from experimentally infected white-footed mice and hamsters (10, 26). To our knowledge, there are no reports of an experimental model using outbred mice. A major advantage of outbred mice is their cheaper price. The present study was designed to establish an experimental model with outbred ddY mice and to evaluate the usefulness of this model by passive and active immunization experiments. Three- and 10-week-old outbred ddY mice and 4-week-old golden hamsters were purchased from SLC Japan (Hamamatsu, Japan). Animals were housed at five per cage at an ambient temperature of 22°C. The test organism used was strain 297, originally isolated from human spinal fluid (27). The virulent strain, passaged seven times in vitro, has been maintained by passage in hamster and cultured twice in BSKII medium (1) at 34°C. Mice and hamsters were inoculated i.p. with 108 cells of B. burgdorfien. Numbers of spirochetes in cultures were determined by the counting method of Stoenner (28). This challenge dose represents approximately 10,000 100% infectious doses of this spirochete. At 14 days after inoculation, mice were sacrificed, and
TABLE 1. Isolation of B. burgdorferi from experimentally infected ddY mice No. of mice with positive sample/no. of mice testedb
Day" Day5 7 14 28 56 84
~
Hat Heart
4/5 5/5 5/5
4/5 5/5 5/5 3/5 5/5 4/4
5/5 3/5
4/5
Total (% positive)
26/30 (87)
a Days postinoculation. b Mice were injected i.p. with B. *
lddr Bladder
26/29 (90)
Heart or
Hbladder 5/5
5/5 5/5 5/5
Spleen
Kidney
4/5
4/5 3/5
1/5
2/5
0/5
0/5
0/5
1/5 0/5 0/5 0/5 0/5
3/30 (10)
1/30 (3)
0/5
1/5 14/30 (47)
30/30 (100)
Urine
1/5 1/5 0/5
5/5 2/5 1/5 1/5
5/5 5/5
Liver
10/30 (33)
1/5
0/5
Blood
2/5 2/5
0/5
0/5
0/5
1/5 5/30 (7)
burgdorfen (108 cells per mouse) on day zero. After 14 days, mice were sacrificed and tissues were cultured in BSKII medium.
Corresponding author. 3016
VOL. 30, 1992
NOTES
TABLE 2. Susceptibility of outbred ddY mice and hamsters to B. burgdorferi infection No. of cells inoculated
101 102
103 104 105 106 107
108
No. positive/no. of animals used (% positive)' 10-wk-old mice 4-wk-old hamsters
3-wk-old mice 1/5 1/5 4/5 5/5 5/5 5/5 5/5
(20) (20) (80) (100) (100) (100) (100)
5/5 (100)
NT' 0/5 (0) 3/5 (60) 4/5 (80) 5/5 (100) 5/5 (100) 5/5 (100)
5/5 (100)
0/5 (0) 2/5 (40) 0/5 (0) 5/5 (100) 4/5 (80) 5/5 (100) 5/5 (100)
5/5 (100)
"Mice and hamsters were challenged i.p. with B. burgdorferi on day zero. After 14 days, animals were sacrificed and heart and urinary bladder were cultured in BSKII medium. b NT, not tested.
blood, urine, hearts, bladders, kidneys, livers, and spleens were cultured in BSKII medium for 4 weeks by the method of Schwan et al. (26). Efficiencies of cultivation of B. burgdorferi 297 from the heart (87%) and the bladder (90%) were significantly higher than that from spleen, kidney, liver, urine, and blood through 5 to 84 days after inoculation of strain 297 (Table 1). Furthermore, isolation efficiency from the heart and the urinary bladder combined was 100% at all dissection days. To determine susceptibility of ddY mice to infection with B. burgdorferi, 3- and 10-week-old ddY mice and 4-week-old hamsters were challenged i.p. At 14 days postinoculation, animals were sacrificed and the heart and the urinary bladder were cultured in BSKII medium. B. burgdorfen was cultured from the heart and/or the bladder of all 3- and 10-week-old mice inoculated with 104 cells, 105 cells, or greater than 105 cells (Table 2). Hamsters inoculated with 104 cells or more were also infected. These results suggest that the sensitivity of outbred ddY mice to infection with B. burgdorferi was much the same as that of hamsters. In the passive immunization experiment, 100 ,ul of undiluted or diluted anti-297 immune rabbit serum or normal rabbit serum was inoculated i.p. into 3-week-old mice on day zero. Preparations of immune rabbit serum were described previously (17). After 20 h, the mice were challenged i.p. with 108 B. burgdorferi cells. Outbred ddY mice receiving passively transferred undiluted and 10-fold-diluted immune serum were completely protected from B. burgdorfen infection (Table 3). On the other hand, immune serum diluted 100-fold or greater and normal rabbit serum could not TABLE 3. Passive immunization of ddY mice with immune rabbit serum" Serum and dilution
Anti-297 Undiluted 1:10 1:100 1:1,000 1:10,000 1:100,000 Normal, undiluted
of mice used
% Protection
0/5 0/5
100 100
5/5 5/5 5/5 5/5 5/5
0 0 0 0 0
a Mice were inoculated i.p. with immune or normal rabbit serum. After 20 h, mice were challenged i.p. with B. bwgdorferi (108 cells per mouse). After 14 days, the heart and urinary bladder were removed from each mouse and cultured in BSKII medium.
3017
TABLE 4. Active immunization of ddY mice with lyophilized
whole cells of B. Dose of vaccine
(AIg [dry wt]/mouse) 10 25 50 100 0 (saline)
burgdorferfi"
No. positive/no. of mice used (% protection) 14 days
28 days
1/5 (80) 1/5 (80) 0/5 (100) 0/5 (100) 5/5 (0)
1/5 (80) 1/5 (80) 0/5 (100) 0/5 (100) 4/4 (0)
aMice were immunized i.p. with lyophilized whole cells of B. burgdorfen, and 14 or 28 days postimmunization, mice were challenged i.p. with B. burgdorferi (108 cells per mouse). After 14 days, heart and urinary bladder tissues were removed from each mouse and cultured in BSKII medium.
prevent infection. In active immunization experiments, lyophilized B. burgdorferi cells were resuspended in saline at concentrations of 100, 250, 500, and 1,000 ,ug/ml. Threeweek-old mice were given a single dose (100 ,ul per mouse) of the vaccine i.p. The mice were challenged i.p. with 108 B. burgdorferi cells at 14 and 28 days after vaccination. On day 14 after challenge, mice were sacrificed 4nd the heart and the urinary bladder were removed and cultured in BSKII medium. i.p. of 10 and 25 ,ug of vaccine protected four of five mice from challenge with 108 cells of B. burgdorferi. Increasing the amount of vaccine to 50 or 100 ,ug elicited a 100% protective response in mice at both postvaccination times. All nine control mice receiving saline injections were culture positive (Table 4). In this study, we evaluated the usefulness of outbred mice as an experimental model for B. burgdorferi infection. We demonstrated that the heart and the urinary bladder of infected mice were excellent sources of B. burgdorferi. On the other hand, isolation from the liver, the blood, and the urine was less productive. This observation is in accord with the results reported previously (10, 26). Especially worthy of notice is that B. burgdorferi was isolated from the heart and/or the urinary bladder of all infected mice. Since this result suggested that cultivation of the heart and the bladder was sufficient to estimate infection in this model, cultivation was restricted to the two organs in the following experiments. Histopathological changes related to Lyme disease were not displayed by the ddY mice except for slight lymphocytic infiltration in the heart (data not shown). Minor histological changes were reported in hamsters (8-10), normal C.B.-17 mice (22), and the hispid cotton rat (8). We believe that these observations are related to the rodents being primarily reservoir hosts for this spirochete in nature. The spirochete was cultured in urine sample from 1 of 30 mice tested. To our knowledge, this is the first report of isolating B. burgdorferi from urine of experimentally infected mice. Sensitivity of mice to infection with strain 297 was much the same as that for hamsters. Their 100% infectious doses were 104 to 105 cells per animal by i.p. inoculation. The sensitivity to infection of the outbred ddY mice was very similar to that of hamnsters and rats reported by other researchers (13, 19). Furthermore, in the passive and active immunization experiments, a dose-dependent protective effect in mice was exhibited. In conclusion, these results indicate that the outbred ddY mice model is very useful and effective for evaluating the effectiveness of vaccination.
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NOTES
This study was supported in part by a Grant-in-Aid for Encouragement of Young Scientists no. 02857348 and a Grant-in-Aid for Scientific Research no. 03807025 from the Ministry of Education, Science, and Culture of Japan and Public Health Service grant AI 29739. REFERENCES 1. Barbour, A. G. 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J. Biol. Med. 57:521-525. 2. Barthold, S. W. 1991. Infectivity of Borrelia burgdorfeni relative to route of inoculation and genotype in laboratory mice. J. Infect. Dis. 163:419-420. 3. Barthold, S. W., D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody. 1990. Lyme borreliosis in selected strains and ages of laboratory mice. J. Infect. Dis. 162:133-138. 4. Barthold, S. W., K. D. Moody, and D. S. Beck. 1990. Susceptibility of laboratory rats to isolates of Borrelia burgdorferi from different geographic areas. Am. J. Trop. Med. Hyg. 42:596-600. 5. Barthold, S. W., K. D. Moody, G. A. Terwilliger, P. H. Duray, R. 0. Jacoby, and A. C. Steere. 1988. Experimental Lyme arthritis in rats infected with Borrelia burgdorferi. J. Infect. Dis. 157:842-846. 6. Barthold, S. W., D. H. Persing, A. L. Armstrong, and R. A. Peeples. 1991. Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139:263-273. 7. Burgdorfer, W. 1984. The New Zealand White rabbit: an experimental host for infecting ticks with Lyme disease spirochetes. Yale J. Biol. Med. 57:609-612. 8. Burgdorfer, W., and K. L. Gage. 1987. Susceptibility of the hispid cotton rat (Sigmodon hispidus) to the Lyme disease spirochete (Borrelia burgdorferi). Am. J. Trop. Med. Hyg. 37:624-628. 9. Duray, P. H., and R. C. Johnson. 1986. The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorfen (42251). Proc. Soc. Exp. Biol. Med. 181:263-269. 10. Goodman, J. L., P. Jurkovich, C. Kodner, and R. C. Johnson. 1991. Persistent cardiac and urinary tract infections with Borrelia burgdorfen in experimentally infected Syrian hamsters. J. Clin. Microbiol. 29:894-896. 11. Greene, R. T., R. L. Walker, W. L. Nicholson, H. W. Heidner, J. F. Levine, E. C. Burgess, M. Wyand, E. B. Breitschwerdt, and H. A. Berkhoff. 1988. Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorfen) in experimentally and naturally exposed dogs. J. Clin. Microbiol. 26:648-653. 12. Johnson, R. C., C. Kodner, and M. Russell. 1986. Active immunization of hamsters against experimental infection with Borrelia burgdorferi. Infect. Immun. 54:897-898. 13. Johnson, R. C., C. Kodner, and M. Russell. 1986. Passive immunization of hamsters against experimental infection with the Lyme disease spirochete. Infect. Immun. 53:713-714. 14. Johnson, R. C., N. Marek, and C. Kodner. 1984. Infection of Syrian hamsters with Lyme disease spirochetes. J. Clin. Micro-
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