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Susceptibility of the black-legged tick, Ixodes scapularis, to the Lyme disease spirochete, Borrelia burgdorferi
Zbl. Bakt. Hyg. A 263, 15-20 (1986)
Animal Experiments and Ecology Susceptibility of the black-legged Tick, Ixodes scapularis, to the Lyme Disease Spirochete, Borrelia burgdorferi WILL Y BURGDORFER J and KENNETH L. GAGE 2 I
2
Department of Health and Human Services, Public Health Service, National Institutes of Health, National Insnture of Allergy and Infectious Diseases, Laboratory of Pathobiology, Rocky Mountain Laboratories, Hamilton, Montana 59840 Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019
Summary Experiments to determine whether Ixodes scapularis can be infected wirh the Lyme disease agent, Borrelia burgdorferi, demonstrated that infection and transstadial transmission occurs In up to 73 % of larval ticks that had fed on spirochetemic rabbits. In a hrnired number of nymphal ticks examined, the spirochetes were found only in the midgut. Feeding nymphal 1.scapularis on a normal rabbit resulted in blood infection characterized by two distinct periods of spirochetemia, suggesting the occurrence of a relapse phenomenon similar to that in tick-borne relapsing fevers. This was also indicated by the percentage of infected ticks recovered daily during the experiment. Accordingly, ticks fed during low spirocheremias or negative blood phase, showed low infection rates or were not infected whereas those fed during peak spirochetemias had high infection rates. Of 11 adult 1. scapularis examined to date, 6 were infected but the spirochetes were restricted to the midgut. These preliminary findings establish the susceptibility of I. scapularis to B. burgdorferi and the potential role of this tick as an efficient vector of the Lyme disease spirochete.
Introduction
In the United States, the" Ixodes ricinus complex" of ticks is represented by three species, namely Darnmin's northeastern deer tick, Ixodes dammini, the western blacklegged tick, Ixodes pacificus, and the black-legged tick, Ixodes scapularis. Both, 1. dammini and 1. paaficus are recognized as the main tick vectors of the Lyme disease spirochete, Borrelia burgdorferi, within their geographical distribution areas. Ixodes dammini for which infection rates up to 100% have been recorded for foci in the state of New York (Burgdorfer and Benach, unpublished), extends from the southern most area of Delaware northward, primarily in coastal areas through Pennsylvania, New Jersey, New York, Connecticut, Rhode Island, and Massachusetts. It is also found in southern Ontario, Canada, and in the north central states of Wisconsin and Minnesota.
16
W. Burgdorfer and K. L. Gage
Ixodes pacificus is found west of the Cascade and Sierra Nevada mountain ranges, from British Columbia southward to Baja, California and Mexico. It also occurs in Nevada and west of the Wasatch Range in Utah, and there is a single report from Idaho. In a preliminary tick/spirochete survey conducted in southwest Oregon and northern California, 2 percent or less of I. pacificus were infected with spirochetes indistinguishable from B. burgdorferi (4). The third member of the American" Ixodes ricinus complex", Ixodes scapularis, has the widest geographical distribution. It is found from Florida westward through the Gulf Coast states to Texas. From there it extends northward through Oklahoma, Kansas, Missouri, and Iowa. It also occurs in Ohio, southern Indiana, Tennessee, Arkansas, and in the coastal states of Georgia, and North and South Carolina. The increased number of Lyme disease cases reported from these areas (5) suggests that this tick (of which all stages, but particularly adults, will readily feed on man), may also be involved in the epidemiology and ecology of Lyme disease. This speculation induced us to experimentally evaluate the susceptibility of I. scapularis to the Lyme disease spirochete, Borrelia burgdorferi. Materials and Methods Larval I. scapularis were allowed to feed on New Zealand white rabbits that had been exposed to B. burgdorferi. The methods were similar to those described previously for the experimental infection of I. dammini, I. pacificus, and I. ricinus (3). Briefly, larval I. scapularis derived from females collected near Stillwater, Oklahoma, were fed on 5 to 8-week old rabbits infected with the Lyme disease spirochete. Before use, each rabbit was tested by indirect immunofluorescence for antibodies to B. burgdorferi and was found to be negative. Similarly, only larval I. scapularis that came from females free of microscopically detectable pathogens were used. Infection was initiated in two rabbits (Nos 3848, 3849), by intravenous injection of 0.5 ml of a suspension prepared by triturating the tissues of four spirochete-infected I. dammini females from Shelter Island, New York, in 3 ml of 1/15 M phosphate buffered saline, pH 7.0. Beginning on day 8 after inoculation, and at 3-day intervals thereafter, several hundred larval I. scapularis were placed freely on each rabbit contained in a wire cage over a tray of water to prevent escape of ticks that would not feed, and to collect repleted ticks that had dropped. Engorged larvae were collected daily and were stored in cotton-stoppered vials at 90 to 100 percent relative humidity. About 6 to 10 weeks after the ticks had molted to nymphs, 10 to 36 specimens for each dropoff day were dissected and evaluated for spirochetal infections by direct fluorescent antibody staining of tissue smears. For this purpose, sera of New Zealand white rabbits that had been immunized with B. burgdorferi (isolate B 31) were labeled with fluorescein-isothiocyanate according to Peacock et al. (6). In a single transmission experiment, 100 I. scapularis nymphs that had fed as larvae on rabbit No 3849, were allowed to feed on a normal rabbit. Beginning day 8 after placement of ticks, and every other day thereafter until day 30, 0.5 ml of blood drawn from the rabbit's ear vein was inoculated in aliquots of 0.25 ml into each of two tubes containing BSK II medium (1). These tubes were then incubated at 34°C for 4 weeks during which time they were periodically examined for spirochetes by darkfield microscopy.
Results The feeding of larval I. scapularis on New Zealand white rabbits experimentally infected with B. burgdorferi resulted in spirochetal infections that were maintained
Infection of I. scapularis with B. burgdorferi
17
R No. 3848 for infection of larval Ixodes scapularis with Borrelia burgdorferi
'\,
70
t ••
60
t
50 40
9/26
9/25
21
23
30 7 /36
...
20 10 -
2 /15
1/20
5
11
9
'3
15
17
19
25
DAYS
Figure 1: New Zealand white rabbit used to infect larval Ixodes scapularis with Borrelia burg-
dorferi Normal I. scapularis larvae placed Engorged I. scapularis larvae off "*" Number of infected ticks over number of ticks examined
R No. 3849 for infection of larval Ixodes scapularis with Borrelia burgdorferi
'\,
80 8 /1 1
1 1/1 5
70
' 3/ 20
60 5/10
50 40 30 20 1/ 20 1/20
10 1/3 5 0/1 4 1/ 2 7 0/ 1 • • • 0 /2 0 5
1 \
13
15
.7
•• '9
21
23
25
DA YS
Figure 2: New Zealand wlute rabbit used to infect larval Ixodes scapularis with Borrelia
burgdorferi ,. Normal I. scapularis larvae placed "', Engorged I. scapularis larvae off "* ,. Number of infected ticks over number of ticks examined 2 ZbL Bake Hyg. A 261 1-2
18
W. Burgdorfer and K. L. Gage
transstadially and were detectable microscopically in nymphal ticks. For rabbit No 3848, the highest infection rate of 36 percent was recorded for ticks that had completed engorgement on the 23rd day after inoculation of the host animal. On the other hand, feeding on rabbit No 3849, resulted in peak infection rates of 73 and 72 percent of I. scapularis that had dropped as larvae on days 17 and 23, respectively. An analysis of Figures 1 and 2, which summarize the percentages of infected I. scapularis nymphs per each drop-off day for both rabbits, indicates the occurrence of spirochetemias characterized by alternating low and high concentrations of spirochetes. Thus, the spirochetemia in rabbit No 3848 was of sufficient concentration to infect few ticks by day 11, increased by day 14 to infect 19 percent of ticks, but then decreased gradually to produce few or no infected ticks until about day 20 when the number of spirochetes increased again to infect 30 to 36 percent of ticks. An even more typical "relapse phenomenon" with three peaks of spirochetemia was recorded for rabbit No 3849. After an initial spirochetemia sufficient to infect at least 1 of 35 I. scapularis off on day 11, the rabbit became negative or had spirochetes in concentrations insufficient to infect ticks that had completed engorgement on days 12, 13, and 14. A 73 percent infection rate recorded for ticks off on day 17 reflects the reappearance of spirochetes in much greater concentrations. This peak was then followed by decreasing numbers of spirochetes that resulted in only 5 percent infection rates for ticks off on days 18 and 19. Infection rates of 65,50, and 72 percent for ticks that had dropped on days 21, 22, and 23, respectively, indicate the occurrence of a third peak of spirochetemia with increased numbers of spirochetes. Of 25 spirochete-infected 1. scapularis nymphs examined individually to determine distribution of B. burgdorferi in their tissues, all exhibited midgut infections only. Placement of 100 I. scapularis nymphs that had completed engorgement as larvae on rabbit No 3849 on days 16 and 17, resulted in the feeding of 99 ticks and in the transmission of spirochetes to normal rabbit No 1583. Blood samples from this animal cultured in BSK II medium, were positive for B. burgdorferi on day 8, negative on days 10 and 12, again positive on days 14 and 16, but negative on day 18 and thereafter. Lastly, of the nymphal ticks that had fed on rabbit No 1583, 11 so far have molted to adults and were examined for spirochetes. Six were infected with spirochetes in their midgut but not in other tissues.
Discussion The results of these experiments indicate the susceptibility of the black-legged tick, I.
scapularis, to the Lyme disease spirochete, B. burgdorferi and establish this tick's potential role as a vector in the south central and southeastern U.S. where sporadic cases of Lyme disease have occurred. Spirochetal infections in nymphs and adults have so far been limited to midgut tissues. Thus, the development of the spirochete in I. scapularis appears similar to that in I. dammini and I. pacificus in the U.S., and in I. ricinus in Europe. Future examination of additional adult I. scapularis from the present study undoubtedly will provide more detailed information about the development of B. burgdorferi in this tick. Spirocheternias of sufficient concentrations to infect up to 30 percent of simultaneously feeding larval I. dammini, 1. pacificus, or I. ricinus have occurred in New Zealand white rabbits fed on by mfected ticks or inoculated with tick tissues infected with B. burgdorferi (3). Although in that study no reference was made to the occurrence of
Infection of I. scapularis with B. burgdorferi
19
more than one peak in spirochetal concentrations in the blood, variations in the percentage of infected ticks for each drop-off day suggested spirochetemias with alternating high and low levels of spirochetes. This phenomenon - characteristic for tick-borne relapsing fevers - again became apparent in the present study in which both rabbits (Nos 3848, 3849) had an initial peak concentration of spirochetes followed by at least one relapse. A review, of an hitherto unpublished experiment with I. dammini (see Figure 3), suggests the occurrence within 14 days of two relapses with spirocheta l concentrations sufficient to infect 50 percent or more ticks at the height of spirochetemias.
R No. 385 1 for infection of larval Ixodes dammini with Borrelia burgdorferi
9
I.
.3
.5
.7
•9
2•
23
25
DA YS
Figure 3 : Ne w Zealand wh ite rabb it used to infect larva l Ixodes dammini with Borrelia
burgdorjeri e- No rma l l. damm ini lar vae placed * * engor ged l. dammini lar vae of f
" ** N umber of infected ticks over nu mber of ticks exa mined.
Additional evidence of a "relapse phenomenon" was indicated by the alternating positive and negative blood cultures from rabbit No 1583 that had been fed on by infected I. scapularis nymphs. Relapsing spirochetemias have also been observed in the cotton rat (Sigmodon hispidus) infected with B. burgdorferi (Burgdorfer, Gage and Barbour - unpublished data ). Recently, this phenomenon has been shown (Barbour, Burgdorfer, and Gage, unpublished data) to be related to antigenic variations similar to those described for the tick-borne relapsing fever spirochete B. hermsii (7, 2). Acknowledgement We gra tefully ackn owledge Dr. Robert Barker, Okl ah om a State Univer sity, Department of Entom ology, Stillwater, O klahoma for supplying the ticks used in th is study.
20
W. Burgdorfer and K. L. Gage
References 1. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochetes. Yale]. BioI. Med . 57
(1984) 521-525 2. Barbour, A. G., S. L. Tessier, and H. G. Stoenner: Variable major proteins of Borreliahermsii. J. Exp. Med. 156 (1982) 1312-1 324 3. Burgdorfer, W.: The New Zeal and white rabbit: an experimental host for infecting ticks with Lyme disease spirochetes. Yale] . BioI. Med . 57 (1984) 609612 4. Burgdorfer, W., S. R. Lane, A. G. Barbour, R. A. Gresbrink, and T. R. Anderson: The western black -legged tick, Ixodes pacificus: a vecto r of Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 34 (1985) 925-930 5. Morbidity and Mortali ty Weekly Report. Centers for Disease Co ntro l. 34 (1985) 376-384 6. Peacock, M., W. Burgdorjer, and R. A. Ormsbee: Rapid fluorescent- ant ibod y conjugation proced ure. Infect. Immun. 3 (1971) 355- 357 7. Stoenner, H. G., T. Dodd, an d C. Larsen: Antigenic vari ation of Borreliahermsii.]. Exp. Med. 156 (1982) 1297-1311 Dr. Willy Burgdorfer, Rock y Mountain Laboratories, Hamilton, MT 59840, U.S.A.
Animal Experiments and Ecology Susceptibility of the black-legged Tick, Ixodes scapularis, to the Lyme Disease Spirochete, Borrelia burgdorferi WILL Y BURGDORFER J and KENNETH L. GAGE 2 I
2
Department of Health and Human Services, Public Health Service, National Institutes of Health, National Insnture of Allergy and Infectious Diseases, Laboratory of Pathobiology, Rocky Mountain Laboratories, Hamilton, Montana 59840 Department of Zoology, University of Oklahoma, Norman, Oklahoma 73019
Summary Experiments to determine whether Ixodes scapularis can be infected wirh the Lyme disease agent, Borrelia burgdorferi, demonstrated that infection and transstadial transmission occurs In up to 73 % of larval ticks that had fed on spirochetemic rabbits. In a hrnired number of nymphal ticks examined, the spirochetes were found only in the midgut. Feeding nymphal 1.scapularis on a normal rabbit resulted in blood infection characterized by two distinct periods of spirochetemia, suggesting the occurrence of a relapse phenomenon similar to that in tick-borne relapsing fevers. This was also indicated by the percentage of infected ticks recovered daily during the experiment. Accordingly, ticks fed during low spirocheremias or negative blood phase, showed low infection rates or were not infected whereas those fed during peak spirochetemias had high infection rates. Of 11 adult 1. scapularis examined to date, 6 were infected but the spirochetes were restricted to the midgut. These preliminary findings establish the susceptibility of I. scapularis to B. burgdorferi and the potential role of this tick as an efficient vector of the Lyme disease spirochete.
Introduction
In the United States, the" Ixodes ricinus complex" of ticks is represented by three species, namely Darnmin's northeastern deer tick, Ixodes dammini, the western blacklegged tick, Ixodes pacificus, and the black-legged tick, Ixodes scapularis. Both, 1. dammini and 1. paaficus are recognized as the main tick vectors of the Lyme disease spirochete, Borrelia burgdorferi, within their geographical distribution areas. Ixodes dammini for which infection rates up to 100% have been recorded for foci in the state of New York (Burgdorfer and Benach, unpublished), extends from the southern most area of Delaware northward, primarily in coastal areas through Pennsylvania, New Jersey, New York, Connecticut, Rhode Island, and Massachusetts. It is also found in southern Ontario, Canada, and in the north central states of Wisconsin and Minnesota.
16
W. Burgdorfer and K. L. Gage
Ixodes pacificus is found west of the Cascade and Sierra Nevada mountain ranges, from British Columbia southward to Baja, California and Mexico. It also occurs in Nevada and west of the Wasatch Range in Utah, and there is a single report from Idaho. In a preliminary tick/spirochete survey conducted in southwest Oregon and northern California, 2 percent or less of I. pacificus were infected with spirochetes indistinguishable from B. burgdorferi (4). The third member of the American" Ixodes ricinus complex", Ixodes scapularis, has the widest geographical distribution. It is found from Florida westward through the Gulf Coast states to Texas. From there it extends northward through Oklahoma, Kansas, Missouri, and Iowa. It also occurs in Ohio, southern Indiana, Tennessee, Arkansas, and in the coastal states of Georgia, and North and South Carolina. The increased number of Lyme disease cases reported from these areas (5) suggests that this tick (of which all stages, but particularly adults, will readily feed on man), may also be involved in the epidemiology and ecology of Lyme disease. This speculation induced us to experimentally evaluate the susceptibility of I. scapularis to the Lyme disease spirochete, Borrelia burgdorferi. Materials and Methods Larval I. scapularis were allowed to feed on New Zealand white rabbits that had been exposed to B. burgdorferi. The methods were similar to those described previously for the experimental infection of I. dammini, I. pacificus, and I. ricinus (3). Briefly, larval I. scapularis derived from females collected near Stillwater, Oklahoma, were fed on 5 to 8-week old rabbits infected with the Lyme disease spirochete. Before use, each rabbit was tested by indirect immunofluorescence for antibodies to B. burgdorferi and was found to be negative. Similarly, only larval I. scapularis that came from females free of microscopically detectable pathogens were used. Infection was initiated in two rabbits (Nos 3848, 3849), by intravenous injection of 0.5 ml of a suspension prepared by triturating the tissues of four spirochete-infected I. dammini females from Shelter Island, New York, in 3 ml of 1/15 M phosphate buffered saline, pH 7.0. Beginning on day 8 after inoculation, and at 3-day intervals thereafter, several hundred larval I. scapularis were placed freely on each rabbit contained in a wire cage over a tray of water to prevent escape of ticks that would not feed, and to collect repleted ticks that had dropped. Engorged larvae were collected daily and were stored in cotton-stoppered vials at 90 to 100 percent relative humidity. About 6 to 10 weeks after the ticks had molted to nymphs, 10 to 36 specimens for each dropoff day were dissected and evaluated for spirochetal infections by direct fluorescent antibody staining of tissue smears. For this purpose, sera of New Zealand white rabbits that had been immunized with B. burgdorferi (isolate B 31) were labeled with fluorescein-isothiocyanate according to Peacock et al. (6). In a single transmission experiment, 100 I. scapularis nymphs that had fed as larvae on rabbit No 3849, were allowed to feed on a normal rabbit. Beginning day 8 after placement of ticks, and every other day thereafter until day 30, 0.5 ml of blood drawn from the rabbit's ear vein was inoculated in aliquots of 0.25 ml into each of two tubes containing BSK II medium (1). These tubes were then incubated at 34°C for 4 weeks during which time they were periodically examined for spirochetes by darkfield microscopy.
Results The feeding of larval I. scapularis on New Zealand white rabbits experimentally infected with B. burgdorferi resulted in spirochetal infections that were maintained
Infection of I. scapularis with B. burgdorferi
17
R No. 3848 for infection of larval Ixodes scapularis with Borrelia burgdorferi
'\,
70
t ••
60
t
50 40
9/26
9/25
21
23
30 7 /36
...
20 10 -
2 /15
1/20
5
11
9
'3
15
17
19
25
DAYS
Figure 1: New Zealand white rabbit used to infect larval Ixodes scapularis with Borrelia burg-
dorferi Normal I. scapularis larvae placed Engorged I. scapularis larvae off "*" Number of infected ticks over number of ticks examined
R No. 3849 for infection of larval Ixodes scapularis with Borrelia burgdorferi
'\,
80 8 /1 1
1 1/1 5
70
' 3/ 20
60 5/10
50 40 30 20 1/ 20 1/20
10 1/3 5 0/1 4 1/ 2 7 0/ 1 • • • 0 /2 0 5
1 \
13
15
.7
•• '9
21
23
25
DA YS
Figure 2: New Zealand wlute rabbit used to infect larval Ixodes scapularis with Borrelia
burgdorferi ,. Normal I. scapularis larvae placed "', Engorged I. scapularis larvae off "* ,. Number of infected ticks over number of ticks examined 2 ZbL Bake Hyg. A 261 1-2
18
W. Burgdorfer and K. L. Gage
transstadially and were detectable microscopically in nymphal ticks. For rabbit No 3848, the highest infection rate of 36 percent was recorded for ticks that had completed engorgement on the 23rd day after inoculation of the host animal. On the other hand, feeding on rabbit No 3849, resulted in peak infection rates of 73 and 72 percent of I. scapularis that had dropped as larvae on days 17 and 23, respectively. An analysis of Figures 1 and 2, which summarize the percentages of infected I. scapularis nymphs per each drop-off day for both rabbits, indicates the occurrence of spirochetemias characterized by alternating low and high concentrations of spirochetes. Thus, the spirochetemia in rabbit No 3848 was of sufficient concentration to infect few ticks by day 11, increased by day 14 to infect 19 percent of ticks, but then decreased gradually to produce few or no infected ticks until about day 20 when the number of spirochetes increased again to infect 30 to 36 percent of ticks. An even more typical "relapse phenomenon" with three peaks of spirochetemia was recorded for rabbit No 3849. After an initial spirochetemia sufficient to infect at least 1 of 35 I. scapularis off on day 11, the rabbit became negative or had spirochetes in concentrations insufficient to infect ticks that had completed engorgement on days 12, 13, and 14. A 73 percent infection rate recorded for ticks off on day 17 reflects the reappearance of spirochetes in much greater concentrations. This peak was then followed by decreasing numbers of spirochetes that resulted in only 5 percent infection rates for ticks off on days 18 and 19. Infection rates of 65,50, and 72 percent for ticks that had dropped on days 21, 22, and 23, respectively, indicate the occurrence of a third peak of spirochetemia with increased numbers of spirochetes. Of 25 spirochete-infected 1. scapularis nymphs examined individually to determine distribution of B. burgdorferi in their tissues, all exhibited midgut infections only. Placement of 100 I. scapularis nymphs that had completed engorgement as larvae on rabbit No 3849 on days 16 and 17, resulted in the feeding of 99 ticks and in the transmission of spirochetes to normal rabbit No 1583. Blood samples from this animal cultured in BSK II medium, were positive for B. burgdorferi on day 8, negative on days 10 and 12, again positive on days 14 and 16, but negative on day 18 and thereafter. Lastly, of the nymphal ticks that had fed on rabbit No 1583, 11 so far have molted to adults and were examined for spirochetes. Six were infected with spirochetes in their midgut but not in other tissues.
Discussion The results of these experiments indicate the susceptibility of the black-legged tick, I.
scapularis, to the Lyme disease spirochete, B. burgdorferi and establish this tick's potential role as a vector in the south central and southeastern U.S. where sporadic cases of Lyme disease have occurred. Spirochetal infections in nymphs and adults have so far been limited to midgut tissues. Thus, the development of the spirochete in I. scapularis appears similar to that in I. dammini and I. pacificus in the U.S., and in I. ricinus in Europe. Future examination of additional adult I. scapularis from the present study undoubtedly will provide more detailed information about the development of B. burgdorferi in this tick. Spirocheternias of sufficient concentrations to infect up to 30 percent of simultaneously feeding larval I. dammini, 1. pacificus, or I. ricinus have occurred in New Zealand white rabbits fed on by mfected ticks or inoculated with tick tissues infected with B. burgdorferi (3). Although in that study no reference was made to the occurrence of
Infection of I. scapularis with B. burgdorferi
19
more than one peak in spirochetal concentrations in the blood, variations in the percentage of infected ticks for each drop-off day suggested spirochetemias with alternating high and low levels of spirochetes. This phenomenon - characteristic for tick-borne relapsing fevers - again became apparent in the present study in which both rabbits (Nos 3848, 3849) had an initial peak concentration of spirochetes followed by at least one relapse. A review, of an hitherto unpublished experiment with I. dammini (see Figure 3), suggests the occurrence within 14 days of two relapses with spirocheta l concentrations sufficient to infect 50 percent or more ticks at the height of spirochetemias.
R No. 385 1 for infection of larval Ixodes dammini with Borrelia burgdorferi
9
I.
.3
.5
.7
•9
2•
23
25
DA YS
Figure 3 : Ne w Zealand wh ite rabb it used to infect larva l Ixodes dammini with Borrelia
burgdorjeri e- No rma l l. damm ini lar vae placed * * engor ged l. dammini lar vae of f
" ** N umber of infected ticks over nu mber of ticks exa mined.
Additional evidence of a "relapse phenomenon" was indicated by the alternating positive and negative blood cultures from rabbit No 1583 that had been fed on by infected I. scapularis nymphs. Relapsing spirochetemias have also been observed in the cotton rat (Sigmodon hispidus) infected with B. burgdorferi (Burgdorfer, Gage and Barbour - unpublished data ). Recently, this phenomenon has been shown (Barbour, Burgdorfer, and Gage, unpublished data) to be related to antigenic variations similar to those described for the tick-borne relapsing fever spirochete B. hermsii (7, 2). Acknowledgement We gra tefully ackn owledge Dr. Robert Barker, Okl ah om a State Univer sity, Department of Entom ology, Stillwater, O klahoma for supplying the ticks used in th is study.
20
W. Burgdorfer and K. L. Gage
References 1. Barbour, A. G.: Isolation and cultivation of Lyme disease spirochetes. Yale]. BioI. Med . 57
(1984) 521-525 2. Barbour, A. G., S. L. Tessier, and H. G. Stoenner: Variable major proteins of Borreliahermsii. J. Exp. Med. 156 (1982) 1312-1 324 3. Burgdorfer, W.: The New Zeal and white rabbit: an experimental host for infecting ticks with Lyme disease spirochetes. Yale] . BioI. Med . 57 (1984) 609612 4. Burgdorfer, W., S. R. Lane, A. G. Barbour, R. A. Gresbrink, and T. R. Anderson: The western black -legged tick, Ixodes pacificus: a vecto r of Borrelia burgdorferi. Am. J. Trop. Med. Hyg. 34 (1985) 925-930 5. Morbidity and Mortali ty Weekly Report. Centers for Disease Co ntro l. 34 (1985) 376-384 6. Peacock, M., W. Burgdorjer, and R. A. Ormsbee: Rapid fluorescent- ant ibod y conjugation proced ure. Infect. Immun. 3 (1971) 355- 357 7. Stoenner, H. G., T. Dodd, an d C. Larsen: Antigenic vari ation of Borreliahermsii.]. Exp. Med. 156 (1982) 1297-1311 Dr. Willy Burgdorfer, Rock y Mountain Laboratories, Hamilton, MT 59840, U.S.A.