Attempted Transmission of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) (JDI Strain) by Ixodes scapularis (Acari: Ixodidae), Dermacentor variabilis, and Amblyomma americanum STANLEY W. MUKOLWE, A. ALAN KOCAN, ROBERT W. BARKER,* {CATHERINE M. KOCAN, AND G E O R G E L. MURPHY College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078
J. Med. Entomol. 29(4): 673-677 (1992)
KEY WORDS Acari, Borrelia burgdorferi, transmission, ticks
Ixodes are the principal vectors for Borrelia burgdorferi (Burgdorfer et al. 1982, Magnarelli et al. 1986, Anderson 1989). In the United States, laboratory studies have shown that under some conditions, Dermacentor variabilis (Say) and Amblyomma americanum (L.) can acquire the spirochete if allowed to feed on infected mammals; however, the organism appears to be transient in these ticks (Piesman & Sinsky 1988, Mather & Mather 1990). In areas of North America where B. burgdorferi is not hyperendemic, tick vectors have yet to be confirmed. Although isolation of B. burgdorferi from field-collected arthropods from nonhyperendemic areas has been reported (Luckhart et al. 1991, Teltow et al. 1991), infectivity trials for mammals and transmission trials by known tick vectors have not been reported. However, laboratory studies have shown that /. scapularis Say transmits B. burgdorferi to hispid cotton rats (Burgdorfer & Gage 1987), New Zealand white rabbits (Burgdorfer & Gage 1986), and laboratory-reared hamsters (Piesman & Sinsky 1988). As part of an ongoing study designed to determine the ecology of Lyme disease in Oklahoma, laboratory studies were conducted to determine TICKS BELONGING TO THE GENUS
1 Department of Entomology, Oklahoma State University, Stillwater, Okla. 74078.
if immature stages of laboratory-reared A. americanum, D. variabilis, and I. scapularis from Oklahoma could acquire infections from feeding on laboratory-infected rabbits. In addition, we studied whether nymphs that had fed as larvae on infected laboratory-reared rabbits were capable of transmitting B. burgdorferi to susceptible rabbits and if antibody could be detected in rabbit sera. Materials and Methods Experimental Animals and B. burgdorferi Infection Procedures. Male New Zealand white rabbits (4-6 mo old) were inoculated with B. burgdorferi (JDI strain). The inoculum was a first passage reisolate from experimentally infected Peromyscus leucopus. This strain was maintained in BSK II media and by mousemedia passage and was identified as B. burgdorferi using monoclonal antibody H5332 (Barbour et al. 1983). This isolate of B. burgdorferi was chosen for use in this study because it is capable of transstadial transmission to rabbits by some ticks (Burgdorfer & Gage 1986), and at the time this study was initiated, no isolates were available from Oklahoma. Rabbits were infested with immature ticks by placing ticks into nylon stockings fitted onto each of the rabbits' ears. Rabbits then were placed into wire cages (17 by 40 by 20
0022-2585/92/0673-0677S02.00/0 © 1992 Entomological Society of America
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ABSTRACT Laboratory-reared Ixodes scapularis Say, Amblyomma americanum (L.), and Dermacentor variabilis (Say) were fed on New Zealand white rabbits experimentally infected with Borrelia burgdorferi (JDI strain). At repletion, spirochetes could be detected by dark-field microscopy only in I. scapularis. Acquisition rates were 18 and 21%. When previously exposed nymphs of each species were fed on susceptible rabbits, /. scapularis was the only tick of the three species that transmitted B. burgdorferi. When a single rabbit was experimentally infected with B. burgdorferi and infested at 7-d intervals with /. scapularis, A. americanum, D. variabilis, and a second time with /. scapularis, B. burgdorferi was detected again only in cultures from the two groups of /. scapularis. When molted nymphs from each tick species were allowed to feed on susceptible rabbits, spirochetes again were isolated only at necropsy from the rabbits on which the two groups of I. scapularis fed.
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zyme substrate was prepared by diluting 10 ml of sodium acetate buffer (0.5 M, pH 5) + 0.83 ml tetramethyl-benzidine (10 mg/ml) in absolute methanol with distilled water to a final volume of 100 ml, adding hydrogen peroxide (0.1 ml of 30%) just before use. Substrate was added at 100 /xl per well and plates were read at 960 nm using an ELISA reader (Vmax, Molecular Dynamics, Palo Alto, Calif.). Kinetics were run for 1 min. Positive samples were those that showed mean milli-OD values greater than mean + 3 SD of the negative controls. Sera with high and low antibody titers were used as reference sera and test values were calculated using the following formula: Titer = [(test OD — low titer reference OD)/(high titer reference OD — low titer reference OD)] x 100. Indirect fluorescent antibody (IFA) procedures followed those of Rogers et al. (1989). Experimental Design. Three New Zealand white rabbits were inoculated intraperitoneally with 2.6 x 106 B. burgdorferi (JDI) spirochetes. Dosage was determined by counting spirochetes in serially diluted volumes of live cultures. The inoculum was a first passage after reisolation from experimentally infected laboratory-reared P. leucopus. At 10 d postexposure, each rabbit was infested with 1,000 laboratory-reared larvae of either A. americanum, D. variabilis, or I. scapularis. At repletion, 100 of each species were dissected and midguts examined under darkfield microscopy (DFM) for the presence of spirochetes. This was done to determine if spirochetes were acquired by the ticks during feeding. Further evaluation of ticks for spirochete presence was not attempted because we determined that the ability to transmit the organism after molting was a better indication of vector competence than the detection of spirochetes in midgut tissues at the time of molting. Tick tissues that harbored spirochetes at repletion were fixed on slides and IFA evaluations conducted. The remaining ticks from each group were allowed to molt to the nymphal stage. Molted nymphs of each species were allowed to feed on B. burgdorferi seronegative rabbits. Serum was taken from each rabbit for ELISA serology beginning 2 wk before infestation and again every 2 wk for 8-10 wk. Between 8 and 10 wk after infestation with nymphs, rabbits were euthanized and liver, spleen, heart, kidneys, and urinary bladder tissues were cultured in BSK II media after grinding under sterile conditions with a mortar and pestle. Media was routinely evaluated for its ability to support spirochete growth. Before use, each new batch of media was inoculated with several recognized strains of B. burgdorferi (JDI, B 31, and SH 282). Media was not used for isolation attempts until it was capable of maintaining spirochetes. Cultures were examined weekly for 6 wk by DFM before they were discarded. The entire experiment was re-
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cm) suspended above water-filled plastic tubs (60 by 60 by 40 cm). Food and water were available ad libitum. Replete ticks were collected from the bottoms of the tubs. Laboratory Maintenance of Ticks. Laboratoryreared A. americanum, D. variabilis, and /. scapularis were maintained in humidity chambers (90-98% RH at 25°C with a 14-h photophase). All tick species originated from wild-caught females from Oklahoma and have been maintained in the laboratory through numerous life cycles. Tick rearing and handling followed procedures described by Patrick & Hair (1975). Serologic Determinations. The presence of serum antibody in experimentally infected rabbits was detected by an indirect kinetic enzymelinked immunosorbent assay (ELISA) following the procedure of Mukolwe et al. (1992a). The procedure involved growing of B. burgdorferi (JDI) in 50-ml tubes that contained BSK II media at 33°C. After 10 d of incubation, spirochete density reached =10 8 per ml. Tubes were centrifuged at 10,000 x g for 30 min at 4°C and washed three times in 0.01 M phosphatebuffered saline (PBS), pH 7.2. The pelleted bacteria were exposed to gamma irradiation (1.4 Mrad from a 6OCo source). Spirochetes were resuspended in 10 ml of sodium citrate, 1.0 M sodium chloride, and sonicated in a Branson Sonifer Cell Disruptor 200 (VWR Scientific, New Haven, Conn.) in three 1-min pulses. The resulting sonicate was centrifuged at 1,000 x g for 10 min. The supernatant was harvested and its protein concentration determined by the BCA assay method (Pierce Chemical Company, Rockford, 111.). The supernatant was stored at —20°C and later used as antigen to sensitize plates. To sensitize plates, supernatant was thawed and diluted to 2.5 /ig/ml in 0.6 M carbonate buffer, pH 9.6. Flat-bottom 96-well plates were sensitized with 100 fim per well overnight at 4°C. Plates were washed with PBS containing 0.05% Tween-20 (PBS-Tween). Test sera was diluted 1:500 with PBS-Tween and 100 /xl was added to each well in duplicate. Controls on each plate consisted of two known positives and 10 known negatives. To get positive sera, rabbits were injected (Difco Labs, Detroit) with 108 whole dead spirochetes in Freund's complete adjuvant followed by two similar inocula in Freund's incomplete adjuvant 2 and 4 wk after the initial inoculation. Plates were sealed and incubated at 37°C in a water bath for 45 min and later washed in PBS-Tween. Biotinylated goatantirabbit IgG (H+L) antiserum (Vector Laboratories, Burlingame, Calif.) was diluted 1:200 and added to the plates at 100 /x\ per well. The plates were incubated as above and washed with PBSTween. An avidin biotin complex kit (ABC, Vector Laboratories, Burlingame, Calif.) was prepared according to kit directions and added 100 )Ltl per well followed by 45-min incubation. En-
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MUKOLWE ET AL.: ATTEMPTED TRANSMISSION OF B. burgdorferi IN OKLAHOMA 675 Table 1. Detection of infection in laboratory-reared /. scapularis, D. variabilis and A. americanum replete larvae fed on B. burgdorferi experimentally infected rabbits, serologic response in rabbits, and transmission results from rabbits used for tick transmission studies Detection in ticks DFMa FA* /. scapularis D. variabilis A. americanum
18/100rf 21/100 0/100 0/100 0/100 0/100
18/100 21/100 — — —
Detection in rabbits ELISA Isolation0 3/3 3/3 1/3
3/3 3/3 —
0/3 0/3 0/3
—
° DFM, dark field microscopy. h FA, fluorescent antibody test. c Transmission to rabbits as accomplished by tick feeding trials. d Number infected/number examined.
kidneys. No control rabbits (ELISA titers: 6.8 and 4.5, and 4.5 and 3.8 for rabbits on which A. americanum and D. variabilis fed, respectively) or rabbits on which A. americanum nymphs were fed seroconverted (ELISA titer, 5.8 and 3.6), whereas one of three rabbits on which nymphal D. variabilis fed did seroconvert (ELISA titers, 4.1 and 38) (Table 1). However, spirochetes were not recovered from any tissues of this rabbit at necropsy. In the second study, when all tick species were fed successively on the same rabbit, spirochetes were observed by DFM in cultures of I. scapularis made at repletion from both infestation groups but were not seen in cultures of either A. americanum or D. variabilis or in any controls that contained larvae of each species. The rabbit on which all of the experimentally exposed ticks fed seroconverted as determined by ELISA serology (ELISA titer, 40) and spirochetes were successfully cultured from the uriResults nary bladder at postmortem. The uninoculated In total, 18 of 100 (18%) replete larval I. scap- rabbit on which control ticks fed did not seroconularis that had fed on B. burgdorferi-inoculated vert (ELISA titer, 3.8) and spirochetes were not rabbits in the first study and 21 of 100 (21%) in recovered. Of the rabbits on which the different the replicate trial were positive at repletion for molted nymphs had fed, only the two infested spirochetes by DFM. The organisms were con- with /. scapularis seroconverted (ELISA titer, 45 firmed to be B. burgdorferi by IF A and reactivity and 48) and spirochetes were recovered from the with monoclonal antibody H5332. Spirochetes kidneys and urinary bladder of each. The rabbits were not detected in control ticks in either on which the A. americanum and D. variabilis study or in any A. americanum or D. variabilis fed did not seroconvert (ELISA titers, 6.5 and that were examined (Table 1). All inoculated 4.6, respectively) and spirochetes were not rerabbits seroconverted (ELISA titers: 46 and 43, covered from any tissues at necropsy. 48 and 45, 45 and 42 for rabbits on which /. scapularis, A. americanum, and D. variabilis fed, respectively) and spirochetes were recovDiscussion ered at necropsy from the urinary bladder of all rabbits, the kidneys of two rabbits, and the The results of this study indicate that the labspleen of one rabbit. All rabbits on which I. scap- oratory rabbits were susceptible to infection with ularis nymphs were fed seroconverted as deter- B. burgdorferi (JDI strain). These results conmined by ELISA serology (ELISA titers, 48 firm those of Burgdorfer (1984). However, in our and 43), and spirochetes were successfully iso- study, infections initiated by both inoculation lated at necropsy from the urinary bladder and and tick transmission were detected in the sera
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peated with a second group of seronegative rabbits. In both studies, uninfected control ticks were allowed to feed on unexposed rabbits as described for the infected ticks. In a second study designed to determine if infestations on different rabbits by the different tick species had influenced the ability to detect spirochetes, a single seronegative rabbit was inoculated intraperitoneally with 2.6 x 106 spirochetes. At 10 d postexposure, the rabbit was infested with 1,000 uninfected laboratory-reared /. scapularis larvae. Larvae were collected at repletion and divided into two groups. One group of 100 was further divided into groups of 10 and ground under sterile conditions and placed into culture, whereas the second group was placed in a humidity chamber and allowed to molt. At 7-d intervals after repletion of the I. scapularis larvae, 1,000 uninfected laboratory-reared A. americanum, D. variabilis, and a second group of /. scapularis larvae were each allowed to feed on the same rabbit. At repletion, ticks were handled as described previously. After the final tick feeding, the rabbit was euthanized and liver, spleen, heart, kidneys, and urinary bladder tissues were placed into culture. Comparable uninfected control ticks were allowed to feed on a unexposed rabbit and were handled as in the experimental group. Molted nymphs of each species that had fed on the B. burgdorferi-inoculated rabbit were allowed to feed on susceptible seronegative rabbits. Sera was taken from each rabbit beginning 2 wk before infestation and every 2 wk for 8 wk. At 8 wk after infestation with nymphs, rabbits were euthanized and liver, spleen, heart, kidneys, and urinary bladder tissues were placed into media.
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Although these results do not conclusively incriminate /. scapularis as a natural vector for B. burgdorferi, they do suggest the potential for such an occurrence. The recent recovery of B. burgdorferi from wild P. leucopus from Oklahoma (Mukolwe et al. 1992b) confirms the need for additional studies on the isolation and char-
acterization of B. burgdorferi strains from fieldcollected ticks from geographic areas where ticks other than /. dammini and I. pacificus are abundant. Acknowledgment This project was supported in part by Project HR9012, Oklahoma Center for the Advancement of Science and Technology (OCAST) and published as Paper J91056 of the College of Veterinary Medicine, Oklahoma State University.
References Cited Anderson, J. F. 1989. Epizootiology of Borrelia in Ixodes ticks and reservoir hosts. Rev. Infect. Dis. 11, suppl. 6: S1451-S1459. Barbour, A. G., S. L. Tessier & W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41: 795-804. Burgdorfer, W. 1984. The New Zealand whiterabbit: an experimental host for infecting ticks with Lyme disease spirochetes. Yale J. Biol. 57: 609612. Burgdorfer, W. & K. L. Gage. 1986. Susceptibility of the black-legged tick, Ixodes scapularis, to the Lyme disease spirochete, Borrelia burgdorferi. Zbl. Bakt. Hyg. 263: 15-20. 1987. Susceptibility of the hispid cotton rat (Sigmadon hispidus) to the Lyme disease spirochete (Borrelia burgdorferi). Am. J. Trop. Med. Hyg. 37: 624628. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt & J. P. Davis. 1982. Lyme disease—a tick borne spirochetosis? Science 216: 1317-1319. Luckhart, S., G. R. Mullen & J. Wright. 1991. Etiologic agent of Lyme disease, Borrelia burgdorferi, detected in ticks (Acari: Ixodidae) collected from a foci in Alabama. J. Med. Entomol. 28: 652-657. Mather, T. N. & M. E. Mather. 1990. Intrensic competence of three ixodid ticks (Acari) as vectors for the Lyme disease spirochete. J. Med. Entomol. 27: 646-650. Magnarelli, L. A., J. F. Anderson, C. S. Apperson, D. Fish, R. C. Johnson & W. A. Chappell. 1986. Spirochetes in ticks and antibodies to Borrelia burgdorferi in white-tailed deer from Connecticut, New York state, and North Carolina. J. Wildl. Dis. 22: 178-188. Mukolwe, S. W., A. A. Kocan & J. H. Wyckoff III. 1992a. A Lyme disease serological survey in dogs and white-tailed deer in Oklahoma. Ann. N.Y. Acad. Sci. (in press). Mukolwe, S. W., A. A. Kocan, R. W. Barker & G. L. Murphy. 1992b. Isolation of Borrelia burgdorferi from Peromyscus leucopus in Oklahoma. J. Wildl. Dis. 28: 281-283. Patrick, C. D. & J. A. Hair. 1975. Laboratory rearing procedures and equipment for multi-host ticks (Acarina: Ixodidae). J. Med. Ent. 12: 38-390. Piesman, J. & R. J. Sinsky. 1988. Ability of Ixodes scapularis, Dermacentor variabilis and Ambly-
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of the rabbits by ELISA serology, and spirochetes were recovered from tissues at necropsy. Additionally, of tick species commonly found in Oklahoma and used in this study, only /. scapularis acquired B. burgdorferi from infected rabbits and transmitted the spirochete to susceptible rabbits. Burgdorfer & Gage (1986) had previously shown that /. scapularis larvae could acquire infections and transstadially transmit B. burgdorferi to rabbits; however, they did not report on the ability to detect antibody in the rabbits nor the ability to reisolate spirochetes at necropsy. Similar studies by Piesman & Sinsky (1988) and Piesman (1988) indicated that /. scapularis is the only tick that could transmit B. burgdorferi to hamsters, even though spirochetes were initially detected in replete larval A. americanum and D. variabilis. However, actual transmission trials were conducted only with nymphal /. scapularis. Naturally infected A. americanum have been reported from Texas (Teltow et al. 1991), New Jersey (Schulze et al. 1984), and Alabama (Luckart et al. 1991) as determined by IFA. However, tick transmission and infectivity trials for mammals were not attempted. Findings by Piesman & Sinsky (1988) indicated that ticks from different geographic areas may vary in their ability to acquire infections with B. burgdorferi because they were able to detect infections in experimentally exposed A. americanum larvae from Alabama but not in larvae from Texas. Although we could not isolate B. burgdorferi from rabbits on which exposed A. americanum or D. variabilis nymphs had fed, a single rabbit on which D. variabilis fed did seroconvert. The acquisition rate of 18 and 21% in /. scapularis obtained in this study appears to be low when compared with results of Piesman (1988), in which 83% of larval /. scapularis fed on hamsters exposed to B. burgdorferi (JDI strain) were confirmed as infected. Newly molted nymphs were not examined for spirochetes, but the ability to transmit the organism to susceptible rabbits was used instead as a biological indicator of transmission potential. Although spirochetosis in rabbits may alternate between spirochetepositive and spirochete-negative periods (Burgdorfer & Gage 1986), we do not feel that this could account for our results because feeding periods were sufficient in all cases to result in infection of I. scapularis. Additional factors that influenced recovery of spirochetes could include the host used, the route of inoculation, the dosage, and timing of tick feeding.
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omma americanum (Acari: Ixodidae) to acquire, maintain, and transmit Lyme disease spirochetes (Borrelia burgdorferi). J. Med. Entomol. 25: 336339. Piesman, J. 1988. Vector competence of ticks in the southeastern United States for Borrelia burgdorferi. Ann. N.Y. Acad. Sci. 539: 417-418. Rogers, S. J., R. J. Morton & C. A. Baldwin. 1989. A serologic survey of Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi in dogs in Oklahoma. J. Vet. Diag. Invest. 1: 154159.
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Schulze, T. L., G. S. Bowen, E. M. Bosler, M. F. Lakat, W. E. Parkin, R. Altman, B. G. Ormiston & J. K. Shisler. 1984. Amblyomma americanum: a potential vector for Lyme disease in New Jersey. Science 224: 601-603. Teltow, G. J., P. V. Fournier & J. A. Rawlings. 1991. Isolation of Borrelia burgdorferi from arthropods collected in Texas. Am. J. Trop. Med. Hyg. 44: 469474. Received for publication 23 October 1991; accepted 24 February 1992.
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J. Med. Entomol. 29(4): 673-677 (1992)
KEY WORDS Acari, Borrelia burgdorferi, transmission, ticks
Ixodes are the principal vectors for Borrelia burgdorferi (Burgdorfer et al. 1982, Magnarelli et al. 1986, Anderson 1989). In the United States, laboratory studies have shown that under some conditions, Dermacentor variabilis (Say) and Amblyomma americanum (L.) can acquire the spirochete if allowed to feed on infected mammals; however, the organism appears to be transient in these ticks (Piesman & Sinsky 1988, Mather & Mather 1990). In areas of North America where B. burgdorferi is not hyperendemic, tick vectors have yet to be confirmed. Although isolation of B. burgdorferi from field-collected arthropods from nonhyperendemic areas has been reported (Luckhart et al. 1991, Teltow et al. 1991), infectivity trials for mammals and transmission trials by known tick vectors have not been reported. However, laboratory studies have shown that /. scapularis Say transmits B. burgdorferi to hispid cotton rats (Burgdorfer & Gage 1987), New Zealand white rabbits (Burgdorfer & Gage 1986), and laboratory-reared hamsters (Piesman & Sinsky 1988). As part of an ongoing study designed to determine the ecology of Lyme disease in Oklahoma, laboratory studies were conducted to determine TICKS BELONGING TO THE GENUS
1 Department of Entomology, Oklahoma State University, Stillwater, Okla. 74078.
if immature stages of laboratory-reared A. americanum, D. variabilis, and I. scapularis from Oklahoma could acquire infections from feeding on laboratory-infected rabbits. In addition, we studied whether nymphs that had fed as larvae on infected laboratory-reared rabbits were capable of transmitting B. burgdorferi to susceptible rabbits and if antibody could be detected in rabbit sera. Materials and Methods Experimental Animals and B. burgdorferi Infection Procedures. Male New Zealand white rabbits (4-6 mo old) were inoculated with B. burgdorferi (JDI strain). The inoculum was a first passage reisolate from experimentally infected Peromyscus leucopus. This strain was maintained in BSK II media and by mousemedia passage and was identified as B. burgdorferi using monoclonal antibody H5332 (Barbour et al. 1983). This isolate of B. burgdorferi was chosen for use in this study because it is capable of transstadial transmission to rabbits by some ticks (Burgdorfer & Gage 1986), and at the time this study was initiated, no isolates were available from Oklahoma. Rabbits were infested with immature ticks by placing ticks into nylon stockings fitted onto each of the rabbits' ears. Rabbits then were placed into wire cages (17 by 40 by 20
0022-2585/92/0673-0677S02.00/0 © 1992 Entomological Society of America
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ABSTRACT Laboratory-reared Ixodes scapularis Say, Amblyomma americanum (L.), and Dermacentor variabilis (Say) were fed on New Zealand white rabbits experimentally infected with Borrelia burgdorferi (JDI strain). At repletion, spirochetes could be detected by dark-field microscopy only in I. scapularis. Acquisition rates were 18 and 21%. When previously exposed nymphs of each species were fed on susceptible rabbits, /. scapularis was the only tick of the three species that transmitted B. burgdorferi. When a single rabbit was experimentally infected with B. burgdorferi and infested at 7-d intervals with /. scapularis, A. americanum, D. variabilis, and a second time with /. scapularis, B. burgdorferi was detected again only in cultures from the two groups of /. scapularis. When molted nymphs from each tick species were allowed to feed on susceptible rabbits, spirochetes again were isolated only at necropsy from the rabbits on which the two groups of I. scapularis fed.
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zyme substrate was prepared by diluting 10 ml of sodium acetate buffer (0.5 M, pH 5) + 0.83 ml tetramethyl-benzidine (10 mg/ml) in absolute methanol with distilled water to a final volume of 100 ml, adding hydrogen peroxide (0.1 ml of 30%) just before use. Substrate was added at 100 /xl per well and plates were read at 960 nm using an ELISA reader (Vmax, Molecular Dynamics, Palo Alto, Calif.). Kinetics were run for 1 min. Positive samples were those that showed mean milli-OD values greater than mean + 3 SD of the negative controls. Sera with high and low antibody titers were used as reference sera and test values were calculated using the following formula: Titer = [(test OD — low titer reference OD)/(high titer reference OD — low titer reference OD)] x 100. Indirect fluorescent antibody (IFA) procedures followed those of Rogers et al. (1989). Experimental Design. Three New Zealand white rabbits were inoculated intraperitoneally with 2.6 x 106 B. burgdorferi (JDI) spirochetes. Dosage was determined by counting spirochetes in serially diluted volumes of live cultures. The inoculum was a first passage after reisolation from experimentally infected laboratory-reared P. leucopus. At 10 d postexposure, each rabbit was infested with 1,000 laboratory-reared larvae of either A. americanum, D. variabilis, or I. scapularis. At repletion, 100 of each species were dissected and midguts examined under darkfield microscopy (DFM) for the presence of spirochetes. This was done to determine if spirochetes were acquired by the ticks during feeding. Further evaluation of ticks for spirochete presence was not attempted because we determined that the ability to transmit the organism after molting was a better indication of vector competence than the detection of spirochetes in midgut tissues at the time of molting. Tick tissues that harbored spirochetes at repletion were fixed on slides and IFA evaluations conducted. The remaining ticks from each group were allowed to molt to the nymphal stage. Molted nymphs of each species were allowed to feed on B. burgdorferi seronegative rabbits. Serum was taken from each rabbit for ELISA serology beginning 2 wk before infestation and again every 2 wk for 8-10 wk. Between 8 and 10 wk after infestation with nymphs, rabbits were euthanized and liver, spleen, heart, kidneys, and urinary bladder tissues were cultured in BSK II media after grinding under sterile conditions with a mortar and pestle. Media was routinely evaluated for its ability to support spirochete growth. Before use, each new batch of media was inoculated with several recognized strains of B. burgdorferi (JDI, B 31, and SH 282). Media was not used for isolation attempts until it was capable of maintaining spirochetes. Cultures were examined weekly for 6 wk by DFM before they were discarded. The entire experiment was re-
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cm) suspended above water-filled plastic tubs (60 by 60 by 40 cm). Food and water were available ad libitum. Replete ticks were collected from the bottoms of the tubs. Laboratory Maintenance of Ticks. Laboratoryreared A. americanum, D. variabilis, and /. scapularis were maintained in humidity chambers (90-98% RH at 25°C with a 14-h photophase). All tick species originated from wild-caught females from Oklahoma and have been maintained in the laboratory through numerous life cycles. Tick rearing and handling followed procedures described by Patrick & Hair (1975). Serologic Determinations. The presence of serum antibody in experimentally infected rabbits was detected by an indirect kinetic enzymelinked immunosorbent assay (ELISA) following the procedure of Mukolwe et al. (1992a). The procedure involved growing of B. burgdorferi (JDI) in 50-ml tubes that contained BSK II media at 33°C. After 10 d of incubation, spirochete density reached =10 8 per ml. Tubes were centrifuged at 10,000 x g for 30 min at 4°C and washed three times in 0.01 M phosphatebuffered saline (PBS), pH 7.2. The pelleted bacteria were exposed to gamma irradiation (1.4 Mrad from a 6OCo source). Spirochetes were resuspended in 10 ml of sodium citrate, 1.0 M sodium chloride, and sonicated in a Branson Sonifer Cell Disruptor 200 (VWR Scientific, New Haven, Conn.) in three 1-min pulses. The resulting sonicate was centrifuged at 1,000 x g for 10 min. The supernatant was harvested and its protein concentration determined by the BCA assay method (Pierce Chemical Company, Rockford, 111.). The supernatant was stored at —20°C and later used as antigen to sensitize plates. To sensitize plates, supernatant was thawed and diluted to 2.5 /ig/ml in 0.6 M carbonate buffer, pH 9.6. Flat-bottom 96-well plates were sensitized with 100 fim per well overnight at 4°C. Plates were washed with PBS containing 0.05% Tween-20 (PBS-Tween). Test sera was diluted 1:500 with PBS-Tween and 100 /xl was added to each well in duplicate. Controls on each plate consisted of two known positives and 10 known negatives. To get positive sera, rabbits were injected (Difco Labs, Detroit) with 108 whole dead spirochetes in Freund's complete adjuvant followed by two similar inocula in Freund's incomplete adjuvant 2 and 4 wk after the initial inoculation. Plates were sealed and incubated at 37°C in a water bath for 45 min and later washed in PBS-Tween. Biotinylated goatantirabbit IgG (H+L) antiserum (Vector Laboratories, Burlingame, Calif.) was diluted 1:200 and added to the plates at 100 /x\ per well. The plates were incubated as above and washed with PBSTween. An avidin biotin complex kit (ABC, Vector Laboratories, Burlingame, Calif.) was prepared according to kit directions and added 100 )Ltl per well followed by 45-min incubation. En-
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MUKOLWE ET AL.: ATTEMPTED TRANSMISSION OF B. burgdorferi IN OKLAHOMA 675 Table 1. Detection of infection in laboratory-reared /. scapularis, D. variabilis and A. americanum replete larvae fed on B. burgdorferi experimentally infected rabbits, serologic response in rabbits, and transmission results from rabbits used for tick transmission studies Detection in ticks DFMa FA* /. scapularis D. variabilis A. americanum
18/100rf 21/100 0/100 0/100 0/100 0/100
18/100 21/100 — — —
Detection in rabbits ELISA Isolation0 3/3 3/3 1/3
3/3 3/3 —
0/3 0/3 0/3
—
° DFM, dark field microscopy. h FA, fluorescent antibody test. c Transmission to rabbits as accomplished by tick feeding trials. d Number infected/number examined.
kidneys. No control rabbits (ELISA titers: 6.8 and 4.5, and 4.5 and 3.8 for rabbits on which A. americanum and D. variabilis fed, respectively) or rabbits on which A. americanum nymphs were fed seroconverted (ELISA titer, 5.8 and 3.6), whereas one of three rabbits on which nymphal D. variabilis fed did seroconvert (ELISA titers, 4.1 and 38) (Table 1). However, spirochetes were not recovered from any tissues of this rabbit at necropsy. In the second study, when all tick species were fed successively on the same rabbit, spirochetes were observed by DFM in cultures of I. scapularis made at repletion from both infestation groups but were not seen in cultures of either A. americanum or D. variabilis or in any controls that contained larvae of each species. The rabbit on which all of the experimentally exposed ticks fed seroconverted as determined by ELISA serology (ELISA titer, 40) and spirochetes were successfully cultured from the uriResults nary bladder at postmortem. The uninoculated In total, 18 of 100 (18%) replete larval I. scap- rabbit on which control ticks fed did not seroconularis that had fed on B. burgdorferi-inoculated vert (ELISA titer, 3.8) and spirochetes were not rabbits in the first study and 21 of 100 (21%) in recovered. Of the rabbits on which the different the replicate trial were positive at repletion for molted nymphs had fed, only the two infested spirochetes by DFM. The organisms were con- with /. scapularis seroconverted (ELISA titer, 45 firmed to be B. burgdorferi by IF A and reactivity and 48) and spirochetes were recovered from the with monoclonal antibody H5332. Spirochetes kidneys and urinary bladder of each. The rabbits were not detected in control ticks in either on which the A. americanum and D. variabilis study or in any A. americanum or D. variabilis fed did not seroconvert (ELISA titers, 6.5 and that were examined (Table 1). All inoculated 4.6, respectively) and spirochetes were not rerabbits seroconverted (ELISA titers: 46 and 43, covered from any tissues at necropsy. 48 and 45, 45 and 42 for rabbits on which /. scapularis, A. americanum, and D. variabilis fed, respectively) and spirochetes were recovDiscussion ered at necropsy from the urinary bladder of all rabbits, the kidneys of two rabbits, and the The results of this study indicate that the labspleen of one rabbit. All rabbits on which I. scap- oratory rabbits were susceptible to infection with ularis nymphs were fed seroconverted as deter- B. burgdorferi (JDI strain). These results conmined by ELISA serology (ELISA titers, 48 firm those of Burgdorfer (1984). However, in our and 43), and spirochetes were successfully iso- study, infections initiated by both inoculation lated at necropsy from the urinary bladder and and tick transmission were detected in the sera
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peated with a second group of seronegative rabbits. In both studies, uninfected control ticks were allowed to feed on unexposed rabbits as described for the infected ticks. In a second study designed to determine if infestations on different rabbits by the different tick species had influenced the ability to detect spirochetes, a single seronegative rabbit was inoculated intraperitoneally with 2.6 x 106 spirochetes. At 10 d postexposure, the rabbit was infested with 1,000 uninfected laboratory-reared /. scapularis larvae. Larvae were collected at repletion and divided into two groups. One group of 100 was further divided into groups of 10 and ground under sterile conditions and placed into culture, whereas the second group was placed in a humidity chamber and allowed to molt. At 7-d intervals after repletion of the I. scapularis larvae, 1,000 uninfected laboratory-reared A. americanum, D. variabilis, and a second group of /. scapularis larvae were each allowed to feed on the same rabbit. At repletion, ticks were handled as described previously. After the final tick feeding, the rabbit was euthanized and liver, spleen, heart, kidneys, and urinary bladder tissues were placed into culture. Comparable uninfected control ticks were allowed to feed on a unexposed rabbit and were handled as in the experimental group. Molted nymphs of each species that had fed on the B. burgdorferi-inoculated rabbit were allowed to feed on susceptible seronegative rabbits. Sera was taken from each rabbit beginning 2 wk before infestation and every 2 wk for 8 wk. At 8 wk after infestation with nymphs, rabbits were euthanized and liver, spleen, heart, kidneys, and urinary bladder tissues were placed into media.
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JOURNAL OF MEDICAL ENTOMOLOGY
Although these results do not conclusively incriminate /. scapularis as a natural vector for B. burgdorferi, they do suggest the potential for such an occurrence. The recent recovery of B. burgdorferi from wild P. leucopus from Oklahoma (Mukolwe et al. 1992b) confirms the need for additional studies on the isolation and char-
acterization of B. burgdorferi strains from fieldcollected ticks from geographic areas where ticks other than /. dammini and I. pacificus are abundant. Acknowledgment This project was supported in part by Project HR9012, Oklahoma Center for the Advancement of Science and Technology (OCAST) and published as Paper J91056 of the College of Veterinary Medicine, Oklahoma State University.
References Cited Anderson, J. F. 1989. Epizootiology of Borrelia in Ixodes ticks and reservoir hosts. Rev. Infect. Dis. 11, suppl. 6: S1451-S1459. Barbour, A. G., S. L. Tessier & W. J. Todd. 1983. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun. 41: 795-804. Burgdorfer, W. 1984. The New Zealand whiterabbit: an experimental host for infecting ticks with Lyme disease spirochetes. Yale J. Biol. 57: 609612. Burgdorfer, W. & K. L. Gage. 1986. Susceptibility of the black-legged tick, Ixodes scapularis, to the Lyme disease spirochete, Borrelia burgdorferi. Zbl. Bakt. Hyg. 263: 15-20. 1987. Susceptibility of the hispid cotton rat (Sigmadon hispidus) to the Lyme disease spirochete (Borrelia burgdorferi). Am. J. Trop. Med. Hyg. 37: 624628. Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt & J. P. Davis. 1982. Lyme disease—a tick borne spirochetosis? Science 216: 1317-1319. Luckhart, S., G. R. Mullen & J. Wright. 1991. Etiologic agent of Lyme disease, Borrelia burgdorferi, detected in ticks (Acari: Ixodidae) collected from a foci in Alabama. J. Med. Entomol. 28: 652-657. Mather, T. N. & M. E. Mather. 1990. Intrensic competence of three ixodid ticks (Acari) as vectors for the Lyme disease spirochete. J. Med. Entomol. 27: 646-650. Magnarelli, L. A., J. F. Anderson, C. S. Apperson, D. Fish, R. C. Johnson & W. A. Chappell. 1986. Spirochetes in ticks and antibodies to Borrelia burgdorferi in white-tailed deer from Connecticut, New York state, and North Carolina. J. Wildl. Dis. 22: 178-188. Mukolwe, S. W., A. A. Kocan & J. H. Wyckoff III. 1992a. A Lyme disease serological survey in dogs and white-tailed deer in Oklahoma. Ann. N.Y. Acad. Sci. (in press). Mukolwe, S. W., A. A. Kocan, R. W. Barker & G. L. Murphy. 1992b. Isolation of Borrelia burgdorferi from Peromyscus leucopus in Oklahoma. J. Wildl. Dis. 28: 281-283. Patrick, C. D. & J. A. Hair. 1975. Laboratory rearing procedures and equipment for multi-host ticks (Acarina: Ixodidae). J. Med. Ent. 12: 38-390. Piesman, J. & R. J. Sinsky. 1988. Ability of Ixodes scapularis, Dermacentor variabilis and Ambly-
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of the rabbits by ELISA serology, and spirochetes were recovered from tissues at necropsy. Additionally, of tick species commonly found in Oklahoma and used in this study, only /. scapularis acquired B. burgdorferi from infected rabbits and transmitted the spirochete to susceptible rabbits. Burgdorfer & Gage (1986) had previously shown that /. scapularis larvae could acquire infections and transstadially transmit B. burgdorferi to rabbits; however, they did not report on the ability to detect antibody in the rabbits nor the ability to reisolate spirochetes at necropsy. Similar studies by Piesman & Sinsky (1988) and Piesman (1988) indicated that /. scapularis is the only tick that could transmit B. burgdorferi to hamsters, even though spirochetes were initially detected in replete larval A. americanum and D. variabilis. However, actual transmission trials were conducted only with nymphal /. scapularis. Naturally infected A. americanum have been reported from Texas (Teltow et al. 1991), New Jersey (Schulze et al. 1984), and Alabama (Luckart et al. 1991) as determined by IFA. However, tick transmission and infectivity trials for mammals were not attempted. Findings by Piesman & Sinsky (1988) indicated that ticks from different geographic areas may vary in their ability to acquire infections with B. burgdorferi because they were able to detect infections in experimentally exposed A. americanum larvae from Alabama but not in larvae from Texas. Although we could not isolate B. burgdorferi from rabbits on which exposed A. americanum or D. variabilis nymphs had fed, a single rabbit on which D. variabilis fed did seroconvert. The acquisition rate of 18 and 21% in /. scapularis obtained in this study appears to be low when compared with results of Piesman (1988), in which 83% of larval /. scapularis fed on hamsters exposed to B. burgdorferi (JDI strain) were confirmed as infected. Newly molted nymphs were not examined for spirochetes, but the ability to transmit the organism to susceptible rabbits was used instead as a biological indicator of transmission potential. Although spirochetosis in rabbits may alternate between spirochetepositive and spirochete-negative periods (Burgdorfer & Gage 1986), we do not feel that this could account for our results because feeding periods were sufficient in all cases to result in infection of I. scapularis. Additional factors that influenced recovery of spirochetes could include the host used, the route of inoculation, the dosage, and timing of tick feeding.
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omma americanum (Acari: Ixodidae) to acquire, maintain, and transmit Lyme disease spirochetes (Borrelia burgdorferi). J. Med. Entomol. 25: 336339. Piesman, J. 1988. Vector competence of ticks in the southeastern United States for Borrelia burgdorferi. Ann. N.Y. Acad. Sci. 539: 417-418. Rogers, S. J., R. J. Morton & C. A. Baldwin. 1989. A serologic survey of Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi in dogs in Oklahoma. J. Vet. Diag. Invest. 1: 154159.
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Schulze, T. L., G. S. Bowen, E. M. Bosler, M. F. Lakat, W. E. Parkin, R. Altman, B. G. Ormiston & J. K. Shisler. 1984. Amblyomma americanum: a potential vector for Lyme disease in New Jersey. Science 224: 601-603. Teltow, G. J., P. V. Fournier & J. A. Rawlings. 1991. Isolation of Borrelia burgdorferi from arthropods collected in Texas. Am. J. Trop. Med. Hyg. 44: 469474. Received for publication 23 October 1991; accepted 24 February 1992.
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