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Culturing Borrelia burgdorferi from spleen and kidney tissues of wild-caught white-footed mice, Peromyscus leucopus
ZbI. Bakt. Hyg. A 263, 34-39 (1986)
Culturing Borrelia burgdorferi from Spleen and Kidney Tissues of Wild-caught White-footed Mice, Peromyscus Jeucopus J OH N F . AN DE RSON 1, R USS ELL C. JOHN SO N 2 , L OUI S A . MAGNA REL LI 1 an d FR ED W . H YD E! I
2
Department of Entom ology, The Con necticut Agricultural Experiment Station, New H aven, Co nnecticut, USA Depa rtme nt of Microbiology, University of Min nesota Medical School, Mi nnea po lis, Mi nneso ta, U.S.A.
Summary
=
Borrelia burgdorferi was isolated mos t frequently from tissue of spleen (n = 13) and kidn ey (n 10) and less often from bloo d (n = 5) of wild-caught Peromyscus leucopus. Preval ence of
infectio n tended to be highest at sites whe re Lyme disease was most common (e.g., 5 of 6 mice were posi tive in East H addam, Connecticut). Spiro chetes were not isolated in Danbur y or New H ar tford , areas where this malady is rare . Howev er, in Fairfield, where the disease is also un com mon,4 of 9 mice were infected . Larva l an d nymp hal I. dammini, contai ning borreliae, pa ras itized P. leucopus at all sites where B. burgdorferi was cultu red fro m mice. Borrel iae were also detected in D. variabilis feeding on host s at two of the sites. P. leucopus appears to be an excellent ani mal to identify focal areas of B. burgdorieri.
Introduction
Borrelia burgdorferi (18), th e cau sative agent of Lyme disease ( 12), has been isolat ed from skin, spina l fluid an d th e blood of hu mans (6, 7, 10, 24,25, 29, 32) and fro m th e bloo d, spleen, kidney, eye and liver tissues o f lab orator y-infected hamsters and wildca ught mammals (1, 3-5, 11, 17, 19,20). Recentl y, this spiroc hete has been culture d fro m th e liver of a bird (Anderson, Johnson, Magnarelli, and Hyde, manu script submitted ). Although the early attem pts to isolate thi s spirochete from huma ns, diagn osed with Lyme disease in th e United Sta tes, resulted in few isolat ions (10, 29), recent attempts in Europe have been mor e successful (32) . Similarly, isolations were scarc e fro m wild mammals (5, 11) unt il borreliae were consi stently isolated from spleens and kidn eys of infected rod ent s (1, 17). We report the pr evalence of B. burgdorferi in rodents captured in ar eas of Connecticut where Lyme disease is common or rare. M aterials and Methods Sher ma n box traps baited with peanut butte r, app le and oatmeal were used to cap tu re rodents in the field. After exa mination for and removal of ticks, each animal was sacrificed. Who le blood was drawn from the heart of each animal, and 2 drops (0.02 ml) were inoculated into each of 2 tubes of Barbou r-Stoermer-Kelly (BSK) medium (8) containing 0.15 % agarose (Seakern LE; FMC
Spleen and Kidney Culture of Borrelia
35
Corp., Rockland, Maine) (17) with or without the reducing compounds L-cysteine HCL (0.023%), DL-dithiothreitol (0.015% ) and superoxide dismutase (0.002%). Animals were surfaced-cleansed with Betadine Surgical Scrubs' and rinsed with 70% isopropyl alcohol. Dissections were performed in a laminar-flow hood where organs were aseprically excised and triturated in 6 ml of BS K medium. AI: 10 dilution of each triturate was cultured in each of 2 tubes of BSK medium containing agarose with or without reducing compounds at 31 °(, Remaining triturated tissues were sent in insulated cartons containing frozen cold packs by overnight courier to the University of Minnesota where duplicate 1: 10 dilutions of the triturates were cultured similarly at 30 °(, Tubes were incubated for 3 weeks at each laborato ry and examined for spirochetes by dark-field microscopy. Monoclonal antibody H5332, which is reactive with the 31,000 molecular weight surface protein of B. burgdorleri (9), was used to confirm the identity of cultured spirochetes. Ticks removed from mice were identified, and 144 were examined for borreliae by direct immunofluorescence with fluorescein isothiocyanare-labeled (FITC) rabbit antibody against B. burgdorferi. Study sites (Fig. 1) in East Haddam and Hebron are areas where the prevalence of Lyme disease in humans is > 149/100,000 population (14). A third site was in Madison where the disease also has been documented (22, 30) but at a lower frequency. Additionally, Danbury and Fairfield, located in western Connecticut, and New Hartford were included to represent areas where prevalence of Lyme disease is low (14).
. 11 /100 ,000
Fig. I. Peromyscus leucopus collecting sites relative to prevalence of documented cases of Lyme disease by county of residence in Connecticut, 1984 (14).
6-12-85 9-13-84 8-14-84 5-29-85 8-28-84 10-31-84
East Haddam Hebron Madison Fairfield Danbury New Hartford
5/6 4/13 2/13 4/9 0/4 0/11
No. of positive mice/no. examined 2/6 0/13 0/13 3/9 0 0
Blood
East Haddam Hebron Madison Fairfield Danbury New Hartford
Collection sites
0
(1.17 ± 1.33) ( .08 ± .28) ( .08 ± .28) ( .67±1.41) 0 0
I.dammini Nymph
(1.17 ± .98) (3.46 ± 4.27) (1.69 ± 3.07) ( .78 ± 1.3) 0
Larva
5/6 3/13 2/13 3/9 0 0
D. uariabilis Nymph
(3.17± 3.31) (.83± .98) .23± .83) 0 0 0 (12.89 ± 21.23) (.44 ± 1.10) ( 1.25 ± .96) 0 0 0
(
Larva
4/6 2/13 1/13 2/9 0 0 3/9
3/9 0
2/9
4/6
3/9
4/6
5/7 1/1 0/1 5/6 -
2/5 6/42 1/19 1/3
-
2/27 0/5
1/17 0/3
0/3
1/5
No. spirochete-positive ticks/ No. ticks examined I. dammini D. variabilis Larva Nymph Larva Nymph
4/6
4/6 2/13
No. of isolations/no. of tissues from different hosts inoculated in: Minnesota Connecticut Right Kidney Left Kidney Spleen Right Kidney Left Kidney Spleen
Mean ± SD ticks per mouse
Table 2. Subadult ticks parasitizing Peromyscus leucopus
Date mice captured
Collection Sites
Table 1. Cultures of Borrelia burgdorferi from blood and tissues of Peromyscus leucopus
>-
e,
~
::l
o""
(1)
::l 0...
';-< ~
"'0\"
Spleen and Kidney Culture of Borrelia
37
Results Borreliae were isol ated fro m P. leucopus captured fro m 4 of 5 sites (T able 1). Prevalence was highest in East H addam, though spiroche tes were iso lated from 2 to 4 mice captured in Hebron, M adison and Fairfield. No sp iroc hetes we re grown in culture from mice trapped in Danb ur y o r N ew Hartford. Organism s culture d fro m each mou se we re identified as B. burgdorferi by th eir reac tion with mon oclon al ant ibo dy (H 5332) . Spirochetes were recovere d fro m spleen and kid ney tissues in bo th Co nnecticut and M inn esot a laboratori es (T able 1). T issues from spleens (n = 13) a nd kidneys (n = 10) yielded more po siti ve cultu res th an did ino culated blo od (n = 5 ). In Co nnecticut, primary cultures were obta ined fro m spleen a nd kidney tissues abo ut as o ften in media containing reducing co mpounds (n = 14 ) as in medi a w itho ut the se che mica ls (n = 16). La rval and nymp hal I. damm ini parasitized P. leucopus in East Haddam, Hebron, M ad ison and Fairfield (Ta ble 2 ). Borr eliae that reacted with FIT C-labeled polyclon al ant ibody to B. burgdorferi w ere detected in I. dammini at each o f th e four study sites an d in D. variabilis colle cted in East Haddam and Fairfi eld .
Discussion Our relatively frequ ent isola tion o f B. burgdorferi fro m spleen and kidney tissue s co nfirms our earlier find ings th at the se bacteria con centrate in th ese o rga ns (1). Since the y are easily and qui ckl y excised, spleens and kidne ys are particul arl y suita ble for de tectio n of this path ogen. Mo re iso lations we re mad e fro m th ese tissues tha n from who le blood. Previous studies have also revea led the per sistence o f B. burgdorferi in liver, br ain , testi cul ar , and eye tissues (1, 17). Inhibition of complem ent , ph agocytosis or high con centrations of urea have been suggested as factors affecting Leptospira growth in kidneys (27) . Leptospira also per sist in br ain tissue s, w hich may be less accessib le to antibo dies (15) . Borrelia burgdorferi appea r to concent ra te in so me of th e sa me tissue s as Leptospira do, po ssibl y for th e sa me reason s. Alth ough Lyme disease has been diagnosed in human s, who reside in several areas o f Co nnecticut, it is most pre valent in 14 towns in th e south -centra l part of the state (14, 22, 30). While we cons isten tly recovered B. burgdorferi fro m P. leucopus captured in are as where this disease is co mmo n, we also discovered infe cted mic e in a region where p reval ence of disease is lowest. Since I. dammini parasit ized th ese ro dent s and since 4 of 9 mice were infected in Fairfield, this community may be a newly developing focus, and human cases may becom e mor e prevalent in the future. We did not isolate these bacteria from mice or co llect I. dammini in Danbury o r N ew H artford, where the disease is rare. Our ability to isol ate the se bo rr eliae fro m the same tissu es in 2 lab or atories 2400 Km ap art ver ifies our earl ier find ings whi ch showed that B. burgdorjeri ca n be cultured from triturated tissue s th at ar e at least 24 hr old (1). Th ese dat a furt her suggest that spiro chetes may be grow n fro m tissue specimens initi ally pro cessed in th e field and later ino culated into culture tu bes in the lab or atory. Ixodes dammini is clearl y th e important vecto r of B. burgdorferi (12 ). Our findin g borreliae in D . variabilis feeding on ro dents is cons istent with earlier work (1, 4 ), but we do no t know if thi s tick can transmi t this pa thogen .
38
J. F. Anderson et al.
In northeastern United States, P. leucopus appears to be an excellent animal to monitor B. burgdorferi infections. It is heavily parasitized by larval and nymphal I. dammini (2, 13,21,23,28), and it has extensive exposure to infected ticks (3-5, 11, 19). It is readily caught at all times of the year and is easily handled both in the field and in the laboratory. Its relatively small home range of less than 1jz hectare (31) ensures that infections were acquired locally. Furthermore, the concentration of spirochetes in different tissues increases the chances for isolation. Cultured strains can be accurately identified serologically (9) or by DNA homology studies (16, 18, 26). Since we have also recovered these bacteria from spleens and kidneys of 2 other feral rodents, Tamias striatus (Eastern chipmunk) (1) and Microtus pennsylvanicus (meadow vole Anderson, Johnson, Magnarelli, and Hyde, manuscript submitted), we suggest that species of rodents in other parts of North America and in other continents may be important reservoirs and that these hosts may be useful for identifying foci.
Acknowledgments We thank CarolLemmon, Clifford Snow III, Patricia Trzcinski, Belinda Rowland, Rose Anne LaBarre, Catherine Hammie, Carrie Kodner, and Marie Russell for their technical assistance. We also acknowledge the support of Public Health Service grants AI 18153 and AM 34744 (awarded to Russel C. Johnson) from the National Institutes of Health. Alan Barbour, Rocky Mountain Laboratory, Hamilton, Montana provided monoclonal antibody H5332.
References 1. Anderson, j. F., R. C. Johnson, L. A. Magnarelli, and F. W. Hyde: Identification of endemic foci of Lyme disease: Isolation of Borrelia burgdorferi from feral rodents and ticks (Dermacentor variabilis). J. Clin. Microbiol. 22 (1985) 36-38 2. Anderson, j. F. and 1. A. Magnarelli: Vertrebrate host relationships and distribution of ixodid ticks (Acari: Ixodidae) in Connecticut, USA. J. Med. Entomol. 17 (1980) 314-323 3. Anderson, j. F. and 1. A. Magnarelli: Spirochetes in Ixodes dammini and Babesia microti on Prudence Island, Rhode Island. J. Infect. Dis. 148 (1983) 1124 4. Anderson, j. F. and L. A. Magnarelli: Avian and mammalian hosts for spirochete-infected ticks and insects in a Lyme disease focus in Connecticut. Yale J. BioI. Med. 57 (1984) 627-641 5. Anderson, j. F., L. A. Magnarelli, W. Burgdorfer, and A. G. Barbour: Spirochetes in Ixodes dammini and mammals from Connecticut. Am J. Trop. Med. Hyg. 32 (1983) 818-824 6. Asbrink, E., B. Hederstedt, and A. Hovmark: The sprirochetal etiology of erythema chronicum migrans Afzelius. Acta. Derm. Venerol. (Stockholm) 64 (1984) 291-295 7. Asbrink, E., B. Hederstedt, and A. Hovmark: The spirochetal etiology of acrodermatitis chronica atrophicans Herxheimer. Acta Derm. Venereal. (Stockholm) 64 (1984) 506-512 8. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochetes. Yale J. BioI. Med. 57 (1984) 521-525 9. Barbour, A. G., S. L. Tessier, and W. j. Todd: Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41 (1983) 795-804 10. Benach, j. L., E. M. Bosler, j. P. Hanrahan, j. L. Coleman, G. S. Habicht, T. F. Bast, D. j. Cameron, j. L. Ziegler, A. G. Barbour, W. Burgdorfer, R. Edelman, and R. A. Kaslow: Spirochetes isolated from the blood of two patients with Lyme disease. N. Eng.
(1983) 740-742 11. Bosler, E. M., j. L. Coleman, j. 1. Benach, D. A. Massey,
.f.
J. Med.
308
P. Hanrahan, W. Burgdorfer,
Spleen and Kidney Culture of Borrelia
39
and A. G. Barbour: Natural distribution of the Ixodes dammini spirochete. Science 220
(1983) 321-322 12. Burgdorfer, W., A. G. Barbour, S. F. Hayes,
13. 14. 15. 16. 17. 18.
J. L. Benach, t: Grunu.aldt, and J. P. Davis: Lyme disease - a tick-borne spirochetosis? Science 216 (1982) 1317-1319 Carey, A. B., W. L. Krinsky, and A. J. Main: Ixodes dammini (Acari: Ixodidae) and associated ixodid ticks in south-central Connecticut, U.S.A. J. Med. Entomol. 17 (1980) 89-99 Checko, P. J.: Lyme disease in Connecticut update. Conn. Health Bull. 98 (1984) 85-88 Hanson, L. E.: Pathogenesis of leptospirosis, p. 295-306. In R. C Johnson (Ed.), The biology of parasitic spirochetes. Academic Press, New York (197 6) Hyde, F. W. and R. C.Johnson: Genetic relationship of Lyme disease spirochetes to Borrelia, Treponema, and Leptospira spp. J. Clin. Microbiol. 20 (1984) 151-154 Johnson, R. C, N. Marek, and C. Kodner: Infection of the Synan hamster With Lyme disease spirochetes. J. Clin. Microbiol. 20 (1984) 1099-11 0 I Johnson, R. C, G. P. Schmid, F. W. Hyde, A. G. Steigerwalt, and D. J. Brenner: Borrelia burgdorferi sp. nov.: etiologic agent of Lyme disease. Int. J. Syst. Bacteriol. 34 (1984)
496-497 19. Levine, J. E, M. L. Wilson, and A. Spielman: Mice as reservoirs of the Lyme disease spirochete. Am. J. Trop. Med. Hyg. 34 1985) 355-360 20. Loken, K. I., C. Wu., R. C. Johnson, and R. F. Bey: Isolation of the Lyme disease spirochete from mammals in Minnesota. Proc. Expt. BIOI. Med. 179 (1985) 300-302 21. Magnarelli, L. A., J. F. Anderson, W. Burgdorfer, and W. Adrian Chappell: Parasitism by Ixodes dammini (Acari: Ixodidae) and antibodies to spirochetes in mammals at Lyme disease foci in Connecticut, U.S.A. J. Med. Entomol. 21 (1984) 52-57 22. Magnarelli, L. A., J. F. Anderson, and W. Adrian Chappell: Geographic distribution of humans, raccoons, and white-footed mice with antibodies to Lyme disease spirochetes in Connecticut. Yale J. BioI. Med. 57 (1984) 619-626 23. Main, A. J., A. B. Carey, M. G. Carey, and R. H. Goodwin: Immature Ixodes dammini (Acari: Ixodidae) on small animals in Connecticut, USA. J. Med. Entomol. 19 \1982)
655-664 24. Pfister, H. W., K. Finbiiupl, V. Preac-Mursic, B. Wilske, and G. Schicrz: The spirochetal
25.
26. 27.
28.
etiology of lymphocytic meningoradicuhtis of Bannwarth lBannwarth's syndrome). J. Neurol. 118 (1984) 1-4 Preac-Mursic, V., B. Wilske, G. Schierz. H. w. Pfister, and K. Einhaupl: Repeated isolations of spirochetes from the cerebrospinal fluid of a patient with meningoradiculitis Bannwarth. Eur. J. Clin. Microbiol. 3 (1984) 564-565 Schmid, G. P.. A. G. Steigerwalt. S. E.Johnson, A. G. Barbour, A. C. Steere, I. At. Robinson, and D. J. Brenner: DNA characterization of the spirochete that causes Lyme disease. J. Chn, Microbiol. 20 (1984) 155-158 Smith, H.: The determinants of microbial pathogenicrty in relation to spirochetal disease, p. 235-247. In: R. C Johnson (Ed., I The biology of parasitic spirochetes. Academic Press, New York (1976) Spielman, A., C. II'I. Clifford, J. Piesman, and M. D. Corwin: Human babesiosis on Nantucket Island, U.S.A.: description of the vector, Ixodes (Ixodes) dammini, n. sp. (Acari: Ixodidae). J. Med. Entomol. 15 (1979) 218-234
29. Steere, A. c. R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista: The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308 (1983) 733-740 30. Steere, A. C. and S. E. Malawista: Cases of Lyme disease in the United States: locations correlated with distribution of Ixodes dammini. Ann. Int. Med. 91 (1979) 730-733 31. Stickel, L. F.: Home range and travels, p. 373-411. In J. A. King (Ed.), Biology of Peromyscus (Rodentia). American Society of Mammalogists (1968) 32. Wilske, B., V. Preac-Mursic, and G. Schierz: 1985. Antigenic heterogeneity of European Borrelia burgdorferi strains isolated from patients and ticks. Lancet 8437 (1985) 1099 Dr. John F. Anderson, Department of Entomology CT. Agr. Exp. Stn. P. O. Box 1106 New Haven, CT 06504, U.S.A. Phone (203) 789-7241
Culturing Borrelia burgdorferi from Spleen and Kidney Tissues of Wild-caught White-footed Mice, Peromyscus Jeucopus J OH N F . AN DE RSON 1, R USS ELL C. JOHN SO N 2 , L OUI S A . MAGNA REL LI 1 an d FR ED W . H YD E! I
2
Department of Entom ology, The Con necticut Agricultural Experiment Station, New H aven, Co nnecticut, USA Depa rtme nt of Microbiology, University of Min nesota Medical School, Mi nnea po lis, Mi nneso ta, U.S.A.
Summary
=
Borrelia burgdorferi was isolated mos t frequently from tissue of spleen (n = 13) and kidn ey (n 10) and less often from bloo d (n = 5) of wild-caught Peromyscus leucopus. Preval ence of
infectio n tended to be highest at sites whe re Lyme disease was most common (e.g., 5 of 6 mice were posi tive in East H addam, Connecticut). Spiro chetes were not isolated in Danbur y or New H ar tford , areas where this malady is rare . Howev er, in Fairfield, where the disease is also un com mon,4 of 9 mice were infected . Larva l an d nymp hal I. dammini, contai ning borreliae, pa ras itized P. leucopus at all sites where B. burgdorferi was cultu red fro m mice. Borrel iae were also detected in D. variabilis feeding on host s at two of the sites. P. leucopus appears to be an excellent ani mal to identify focal areas of B. burgdorieri.
Introduction
Borrelia burgdorferi (18), th e cau sative agent of Lyme disease ( 12), has been isolat ed from skin, spina l fluid an d th e blood of hu mans (6, 7, 10, 24,25, 29, 32) and fro m th e bloo d, spleen, kidney, eye and liver tissues o f lab orator y-infected hamsters and wildca ught mammals (1, 3-5, 11, 17, 19,20). Recentl y, this spiroc hete has been culture d fro m th e liver of a bird (Anderson, Johnson, Magnarelli, and Hyde, manu script submitted ). Although the early attem pts to isolate thi s spirochete from huma ns, diagn osed with Lyme disease in th e United Sta tes, resulted in few isolat ions (10, 29), recent attempts in Europe have been mor e successful (32) . Similarly, isolations were scarc e fro m wild mammals (5, 11) unt il borreliae were consi stently isolated from spleens and kidn eys of infected rod ent s (1, 17). We report the pr evalence of B. burgdorferi in rodents captured in ar eas of Connecticut where Lyme disease is common or rare. M aterials and Methods Sher ma n box traps baited with peanut butte r, app le and oatmeal were used to cap tu re rodents in the field. After exa mination for and removal of ticks, each animal was sacrificed. Who le blood was drawn from the heart of each animal, and 2 drops (0.02 ml) were inoculated into each of 2 tubes of Barbou r-Stoermer-Kelly (BSK) medium (8) containing 0.15 % agarose (Seakern LE; FMC
Spleen and Kidney Culture of Borrelia
35
Corp., Rockland, Maine) (17) with or without the reducing compounds L-cysteine HCL (0.023%), DL-dithiothreitol (0.015% ) and superoxide dismutase (0.002%). Animals were surfaced-cleansed with Betadine Surgical Scrubs' and rinsed with 70% isopropyl alcohol. Dissections were performed in a laminar-flow hood where organs were aseprically excised and triturated in 6 ml of BS K medium. AI: 10 dilution of each triturate was cultured in each of 2 tubes of BSK medium containing agarose with or without reducing compounds at 31 °(, Remaining triturated tissues were sent in insulated cartons containing frozen cold packs by overnight courier to the University of Minnesota where duplicate 1: 10 dilutions of the triturates were cultured similarly at 30 °(, Tubes were incubated for 3 weeks at each laborato ry and examined for spirochetes by dark-field microscopy. Monoclonal antibody H5332, which is reactive with the 31,000 molecular weight surface protein of B. burgdorleri (9), was used to confirm the identity of cultured spirochetes. Ticks removed from mice were identified, and 144 were examined for borreliae by direct immunofluorescence with fluorescein isothiocyanare-labeled (FITC) rabbit antibody against B. burgdorferi. Study sites (Fig. 1) in East Haddam and Hebron are areas where the prevalence of Lyme disease in humans is > 149/100,000 population (14). A third site was in Madison where the disease also has been documented (22, 30) but at a lower frequency. Additionally, Danbury and Fairfield, located in western Connecticut, and New Hartford were included to represent areas where prevalence of Lyme disease is low (14).
. 11 /100 ,000
Fig. I. Peromyscus leucopus collecting sites relative to prevalence of documented cases of Lyme disease by county of residence in Connecticut, 1984 (14).
6-12-85 9-13-84 8-14-84 5-29-85 8-28-84 10-31-84
East Haddam Hebron Madison Fairfield Danbury New Hartford
5/6 4/13 2/13 4/9 0/4 0/11
No. of positive mice/no. examined 2/6 0/13 0/13 3/9 0 0
Blood
East Haddam Hebron Madison Fairfield Danbury New Hartford
Collection sites
0
(1.17 ± 1.33) ( .08 ± .28) ( .08 ± .28) ( .67±1.41) 0 0
I.dammini Nymph
(1.17 ± .98) (3.46 ± 4.27) (1.69 ± 3.07) ( .78 ± 1.3) 0
Larva
5/6 3/13 2/13 3/9 0 0
D. uariabilis Nymph
(3.17± 3.31) (.83± .98) .23± .83) 0 0 0 (12.89 ± 21.23) (.44 ± 1.10) ( 1.25 ± .96) 0 0 0
(
Larva
4/6 2/13 1/13 2/9 0 0 3/9
3/9 0
2/9
4/6
3/9
4/6
5/7 1/1 0/1 5/6 -
2/5 6/42 1/19 1/3
-
2/27 0/5
1/17 0/3
0/3
1/5
No. spirochete-positive ticks/ No. ticks examined I. dammini D. variabilis Larva Nymph Larva Nymph
4/6
4/6 2/13
No. of isolations/no. of tissues from different hosts inoculated in: Minnesota Connecticut Right Kidney Left Kidney Spleen Right Kidney Left Kidney Spleen
Mean ± SD ticks per mouse
Table 2. Subadult ticks parasitizing Peromyscus leucopus
Date mice captured
Collection Sites
Table 1. Cultures of Borrelia burgdorferi from blood and tissues of Peromyscus leucopus
>-
e,
~
::l
o""
(1)
::l 0...
';-< ~
"'0\"
Spleen and Kidney Culture of Borrelia
37
Results Borreliae were isol ated fro m P. leucopus captured fro m 4 of 5 sites (T able 1). Prevalence was highest in East H addam, though spiroche tes were iso lated from 2 to 4 mice captured in Hebron, M adison and Fairfield. No sp iroc hetes we re grown in culture from mice trapped in Danb ur y o r N ew Hartford. Organism s culture d fro m each mou se we re identified as B. burgdorferi by th eir reac tion with mon oclon al ant ibo dy (H 5332) . Spirochetes were recovere d fro m spleen and kid ney tissues in bo th Co nnecticut and M inn esot a laboratori es (T able 1). T issues from spleens (n = 13) a nd kidneys (n = 10) yielded more po siti ve cultu res th an did ino culated blo od (n = 5 ). In Co nnecticut, primary cultures were obta ined fro m spleen a nd kidney tissues abo ut as o ften in media containing reducing co mpounds (n = 14 ) as in medi a w itho ut the se che mica ls (n = 16). La rval and nymp hal I. damm ini parasitized P. leucopus in East Haddam, Hebron, M ad ison and Fairfield (Ta ble 2 ). Borr eliae that reacted with FIT C-labeled polyclon al ant ibody to B. burgdorferi w ere detected in I. dammini at each o f th e four study sites an d in D. variabilis colle cted in East Haddam and Fairfi eld .
Discussion Our relatively frequ ent isola tion o f B. burgdorferi fro m spleen and kidney tissue s co nfirms our earlier find ings th at the se bacteria con centrate in th ese o rga ns (1). Since the y are easily and qui ckl y excised, spleens and kidne ys are particul arl y suita ble for de tectio n of this path ogen. Mo re iso lations we re mad e fro m th ese tissues tha n from who le blood. Previous studies have also revea led the per sistence o f B. burgdorferi in liver, br ain , testi cul ar , and eye tissues (1, 17). Inhibition of complem ent , ph agocytosis or high con centrations of urea have been suggested as factors affecting Leptospira growth in kidneys (27) . Leptospira also per sist in br ain tissue s, w hich may be less accessib le to antibo dies (15) . Borrelia burgdorferi appea r to concent ra te in so me of th e sa me tissue s as Leptospira do, po ssibl y for th e sa me reason s. Alth ough Lyme disease has been diagnosed in human s, who reside in several areas o f Co nnecticut, it is most pre valent in 14 towns in th e south -centra l part of the state (14, 22, 30). While we cons isten tly recovered B. burgdorferi fro m P. leucopus captured in are as where this disease is co mmo n, we also discovered infe cted mic e in a region where p reval ence of disease is lowest. Since I. dammini parasit ized th ese ro dent s and since 4 of 9 mice were infected in Fairfield, this community may be a newly developing focus, and human cases may becom e mor e prevalent in the future. We did not isolate these bacteria from mice or co llect I. dammini in Danbury o r N ew H artford, where the disease is rare. Our ability to isol ate the se bo rr eliae fro m the same tissu es in 2 lab or atories 2400 Km ap art ver ifies our earl ier find ings whi ch showed that B. burgdorjeri ca n be cultured from triturated tissue s th at ar e at least 24 hr old (1). Th ese dat a furt her suggest that spiro chetes may be grow n fro m tissue specimens initi ally pro cessed in th e field and later ino culated into culture tu bes in the lab or atory. Ixodes dammini is clearl y th e important vecto r of B. burgdorferi (12 ). Our findin g borreliae in D . variabilis feeding on ro dents is cons istent with earlier work (1, 4 ), but we do no t know if thi s tick can transmi t this pa thogen .
38
J. F. Anderson et al.
In northeastern United States, P. leucopus appears to be an excellent animal to monitor B. burgdorferi infections. It is heavily parasitized by larval and nymphal I. dammini (2, 13,21,23,28), and it has extensive exposure to infected ticks (3-5, 11, 19). It is readily caught at all times of the year and is easily handled both in the field and in the laboratory. Its relatively small home range of less than 1jz hectare (31) ensures that infections were acquired locally. Furthermore, the concentration of spirochetes in different tissues increases the chances for isolation. Cultured strains can be accurately identified serologically (9) or by DNA homology studies (16, 18, 26). Since we have also recovered these bacteria from spleens and kidneys of 2 other feral rodents, Tamias striatus (Eastern chipmunk) (1) and Microtus pennsylvanicus (meadow vole Anderson, Johnson, Magnarelli, and Hyde, manuscript submitted), we suggest that species of rodents in other parts of North America and in other continents may be important reservoirs and that these hosts may be useful for identifying foci.
Acknowledgments We thank CarolLemmon, Clifford Snow III, Patricia Trzcinski, Belinda Rowland, Rose Anne LaBarre, Catherine Hammie, Carrie Kodner, and Marie Russell for their technical assistance. We also acknowledge the support of Public Health Service grants AI 18153 and AM 34744 (awarded to Russel C. Johnson) from the National Institutes of Health. Alan Barbour, Rocky Mountain Laboratory, Hamilton, Montana provided monoclonal antibody H5332.
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496-497 19. Levine, J. E, M. L. Wilson, and A. Spielman: Mice as reservoirs of the Lyme disease spirochete. Am. J. Trop. Med. Hyg. 34 1985) 355-360 20. Loken, K. I., C. Wu., R. C. Johnson, and R. F. Bey: Isolation of the Lyme disease spirochete from mammals in Minnesota. Proc. Expt. BIOI. Med. 179 (1985) 300-302 21. Magnarelli, L. A., J. F. Anderson, W. Burgdorfer, and W. Adrian Chappell: Parasitism by Ixodes dammini (Acari: Ixodidae) and antibodies to spirochetes in mammals at Lyme disease foci in Connecticut, U.S.A. J. Med. Entomol. 21 (1984) 52-57 22. Magnarelli, L. A., J. F. Anderson, and W. Adrian Chappell: Geographic distribution of humans, raccoons, and white-footed mice with antibodies to Lyme disease spirochetes in Connecticut. Yale J. BioI. Med. 57 (1984) 619-626 23. Main, A. J., A. B. Carey, M. G. Carey, and R. H. Goodwin: Immature Ixodes dammini (Acari: Ixodidae) on small animals in Connecticut, USA. J. Med. Entomol. 19 \1982)
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etiology of lymphocytic meningoradicuhtis of Bannwarth lBannwarth's syndrome). J. Neurol. 118 (1984) 1-4 Preac-Mursic, V., B. Wilske, G. Schierz. H. w. Pfister, and K. Einhaupl: Repeated isolations of spirochetes from the cerebrospinal fluid of a patient with meningoradiculitis Bannwarth. Eur. J. Clin. Microbiol. 3 (1984) 564-565 Schmid, G. P.. A. G. Steigerwalt. S. E.Johnson, A. G. Barbour, A. C. Steere, I. At. Robinson, and D. J. Brenner: DNA characterization of the spirochete that causes Lyme disease. J. Chn, Microbiol. 20 (1984) 155-158 Smith, H.: The determinants of microbial pathogenicrty in relation to spirochetal disease, p. 235-247. In: R. C Johnson (Ed., I The biology of parasitic spirochetes. Academic Press, New York (1976) Spielman, A., C. II'I. Clifford, J. Piesman, and M. D. Corwin: Human babesiosis on Nantucket Island, U.S.A.: description of the vector, Ixodes (Ixodes) dammini, n. sp. (Acari: Ixodidae). J. Med. Entomol. 15 (1979) 218-234
29. Steere, A. c. R. L. Grodzicki, A. N. Kornblatt, J. E. Craft, A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, and S. E. Malawista: The spirochetal etiology of Lyme disease. N. Engl. J. Med. 308 (1983) 733-740 30. Steere, A. C. and S. E. Malawista: Cases of Lyme disease in the United States: locations correlated with distribution of Ixodes dammini. Ann. Int. Med. 91 (1979) 730-733 31. Stickel, L. F.: Home range and travels, p. 373-411. In J. A. King (Ed.), Biology of Peromyscus (Rodentia). American Society of Mammalogists (1968) 32. Wilske, B., V. Preac-Mursic, and G. Schierz: 1985. Antigenic heterogeneity of European Borrelia burgdorferi strains isolated from patients and ticks. Lancet 8437 (1985) 1099 Dr. John F. Anderson, Department of Entomology CT. Agr. Exp. Stn. P. O. Box 1106 New Haven, CT 06504, U.S.A. Phone (203) 789-7241