EXPERIMENTALPARASITOLOGY 70, 55-61 (I!-?%)
Borrelia
burgdorferi and Babe& microti: Efficiency of Transmiss’ion from Reservoirs to Vector Ticks (/iodes dammini) THOMASN.MATHER,'SAM R. TELFORD III, SEAN I. MOORE, AND ANDREW SPIELMAN
Department
of Tropical Public Health. Harvard School of Public Health, Bosion, Massachusetts 02115, U.S.A.
665 Huntington
Avenue,
MATHER, T. N., TELFORD, S. R., III, MOORE,S. I., AND SPIELMAN, A. 1990. Borrelia burgdorferi and Babesia microti: Efftciency of transmission from reservoirs to vector ticks @odes dammini). Experimental Parasitology 70, 55-61. In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically. spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas. e 1990 Academic PESS, IIIC. INDEX DESCRIPTORS: Lyme disease, Borrelia burgdorferi; Babesiosis, Babesia mirroti; Ixodes dammini; Transmission: Peromyscus leucopus.
croti throughout the season of activity of the nymphal tick. The result is nearly universal infection of the reservoir. Estimates of spirochetal prevalence in mice evaluated during several field surveys have demonstrated that about 90% were infected with spirochetes (Anderson et al. 1987a, b; Mather et al. 1989a, b), while Babesia was detected in nearly 80% of the animals sampled during late summer (Etkind et a/. 1980; Spielman et a/. 1981). Therefore, since larval I. dammini must acquire these infectious agents during bloodfeeding, due to the relative lack of inherited infection (Piesman et al. 1986a), their likelihood of encountering a mouse infective for either pathogen would appear to be similar. Paradoxically, despite a high degree of prevalence for both B. burgdorferi and B. microti in reservoir populations, the rate of infection in field-derived vector ticks for these two infectious agents differs.
In the northeastern United States, the white-footed mouse (Peromyscus leucopus) is the. principal reservoir for both the Lyme disease spirochete, Borrelia burgdorferi, and the piroplasm, Babesia microti (Mather et al. 1989b; Spielman et al. 1981). This animal also serves as a major host for immature txodes dammini, the nymphal stage of which is the main tick vector of both infectious agents. Indeed, in nature, nymphal ticks may be concurrently infected with both pathogens (Piesman et al. 1986d), and may transmit both infections simultaneously (Piesman et a/. 1987a). In areas where transmission is intense, mice, as well as other animals, are thus continually inoculated with both B. burgdorferi and B. mi’ Present address: Department of Population Sciences, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 021IS. 55
0014-4894190$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
MATHER ET AL.
Nymphal 1. dammini collected from sites where both pathogens occur in the reservoir have nearly twice the rate of infection for spirochetes as for Babesia (Piesman et al. 1986~). Perhaps some aspect of the reservoir-vector-pathogen dynamics favors spirochetal infection of ticks over their infection by Babesia. Relative efficiencies of pathogen acquisition by ticks, in addition to the success of transstadial passage of their infection, may favor spirochetal infection. Accordingly, we determined the efficiency with which I. dammini, feeding on mice, were infected with either pathogen in laboratory experiments. We measured the prevalence of infection in nymphal ticks previously fed as larvae on these spirochete- or B. microti-infected mice, as well as on mice infected by both pathogens concurrently. MATERIALS
AND METHODS
White-footed mice were from a colony originally derived from several Massachusetts locations, but have been maintained in the laboratory for more than four generations. These mice are free of any natural 8. burgdorferi or 8. microti infection, as determined by tick xenodiagnosis, and examination of blood smears. Mice used for each replicate of the experiment were. in every case but one, age- and sex-matched, and in all but two cases were litter mates. To begin each experiment, mice were infected with B.‘burgdorferi, B. microti, or with both pathogens simultaneously. Animals were infected by allowing either three nymphal I. dammini from a spirocheteinfected cohort or six nymphs from a piroplasminfected cohort to feed to repletion on the mice. To produce concomitant infections, three nymphs from the spirochete-infected cohort were fed on mice simultaneously with six nymphs from the piroplasminfected cohort. The numbers of infecting nymphs used reflected the difference in the cohort infection rate for either pathogen and ensured that each mouse received at least one infectious bite. “Infecting” nymphs were produced by allowing larvae to acquire spirochetes from a mouse previously infected by tick inoculation of the JDI strain of B. burgdorferi (Piesman et al. 1987a)or Babesia from a hamster infected with the GI strain of B. microri (Piesman and Spielman 1982) which has been maintained by alternating tick and syringe passages through golden hamsters. After feeding on the experimental mice, replete infecting nymphs were collected as they detached and were ex-
amined for evidence of infection by either spirochetes or piroplasms, as described below. Three weeks following the initial tick feeding, about 200 larval I. dammini, from egg batches determined to be free of inherited spirochetal infection, were allowed to infest the experimentally infected mice. These larvae were the offspring of field-swept females fed on New Zealand white rabbits in the laboratory. Samples (25-50) of unfed larvae from each egg batch used were examined, without knowledge of the sample’s origin, by a direct fluorescent antibody assay (described below). Larvae engorging on the experimentally infected mice were collected as they detached, were placed in plastic vials and stored in humid chambers at about 95% RH, and were held at 22°C and a l6-hr photophase until nymphs emerged. To determine the prevalence of infection in ticks, nymphs were examined 34 weeks after molting. To detect spirochetes. tick midgut tissues were dissected into a drop of phosphate-buffered saline (pH 7.3) on a glass microscope slide; this tissue was smeared onto the slide with a cover glass, air dried, and fixed in an acetone bath before storage at -20°C. Later, slides were treated with a I:100 dilution of a fluorescein isothiocyanate-conjugated polyclonal antibody prepared from two New Zealand white rabbits inoculated with the Guilford strain of B. burgdorferi (Steere ef al. 1983)and then examined at 400x magnification by fluorescent microscopy. To detect babesial sporozoites, tick salivary glands were similarly dissected from ticks that were prefed on noninfected Syrian golden hamsters for 54 hr (Piesman and Spielman 1982). Glands were allowed to dry onto a gelatin-coated microscope slide, fixed with methanol before storage, and later stained by a modified Feulgens reaction before examination (Piesman et al. 1986~; Blewett and Branagan 1973). In addition, experimentally infected mice were examined for the presence of intraerythrocytic B. microfi infection both before and after tick feeding. Thin blood smears were made from tail snips, dried, fixed in absolute methanol, and stained with IO%,Giemsa at pH 7.0. All slides were examined at 400x magnification for a total of 100 random fields.
RESULTS
We determined whether experimental mice were inoculated with B. burgdorferi, B. microti, or with both infectious agents by examining infecting ticks upon their detachment. In every case, at least one fully engorged nymph, infected with the appropriate agent, was recovered (Table I). Additionally, mice bitten by B. microtiinfecting nymphs all became infected
INFECTION
EFFICIENCIES
OF TICK-BORNE
57
PATHOGENS
rochetes and Bubesiu exhibited infection rates of 82 and 34%, respectively. The spirochetal infection rate was always greater than 75% for those cohorts of ticks feeding on spirochete only infected mice, and was Frequency of usually similar but always greater than 50% infection in for those cohorts feeding on dually infected “infecting” nymphs animals (Table II). These slight differences recovered Kind of Number in infection rates among concurrently inof mice 1 2 3 4 5 6 infection fected nymphs, when compared to nymphs Spirochete IO 433--infected with just one of the pathogens, Piroplasm IO 5 3 1 I 0 0 may not be explained by random variation Concurrent 9 (Fisher’s exact test: spirochete, z, = -3.03, Spirochete 324--P < 0.05; piroplasm, z, = -2.26, P < Piroplasm 2 2 3 2 0 0 0.05). The babesial infection rate among the tick cohorts varied, depending on the aniwithin the 3-week incubation period, as mal tested, although more than two-thirds demonstrated by positive blood smears. No of the cohorts exhibited rates lower than corresponding test exists to detect spiro- 50%. However, one Bubesiu-only infected chetal infection in animals; however, Don- mouse did infect 12 of 15 ticks examined. ahue et al. (1987) demonstrated that mice To assess whether the frequency of inocbecame infective as early as 1 week postin- ulation affected an animal’s infectivity to fectious tick bite. ticks, we compared the number of infected We also determined the prevalence of in- infecting nymphal ticks recovered (Table I) fection in nymphal ticks which had fed as with the pathogen infection rate of tick larvae on these spirochete-, Bubesia-, or cohorts from individual mice (Table II). concurrently infected mice. An average of Correlation coefficients determined for spi92% of nymphs, derived from larvae en- rochete- and piroplasm-infected mice (R’ = gorged on B. burgdor-eri-infected mice, 0.1559; P = 0.667 and R2 = 0.1997; P = proved infected (Table II). In contrast, 0.58, respectively) indicated that the freBabesia sporozoites were detected in only quency of inoculation, within the range of 1 45% of those nymphs derived from larvae to 3 (or 4 in the case of B. microti-infecting feeding on the Bubesiu-infected mice. Ticks nymphs) did not affect an animal’s infectivfrom mice with concurrent infections of spi- ity to ticks. Similar results were obtained TABLE I Frequency of Inoculation of Mice (P. leucopus) Bitten by Nymphal Ticks (I. dammini) Infected with Lyme Spirochetes (B. burgdorferi), Piroplasms (B. microtn, or Both Pathogens
TABLE II Efficiency of Pathogen Acquisition by Ticks (I. dammini) Engorging on Mice (P. leucopus) Infected with Lyme Disease Spirochetes (B. burgdorferi), Piroplasms (B. microti). or with Both Pathogens
Kind of infection Spirochete Piroplasm
Concurrent Spirochete Piroplasm
Number of mice IO IO 9
Distribution of tick cohorts by percent infected
Derived nymphs Number
% infected
~25%
26-50%
.51-75%
215
92% 45%
0 3
0 3
0 3
IO
176 190 159
82% 34%
0 5
0 2
3 2
6 0
~76%
1
58
MATHERETAL.
for mice concurrently infected with spirochetes and piroplasms (R2 = 0.1571; P = 0.687 and R2 =0.2895; P = 0.45, respectively). DISCUSSION
Human cases of Lyme disease are more prevalent than are cases of babesiosis (Dammin ef al. 1981; Dammin 1986). Where both pathogens occur together, the greater risk of acquiring infection associated with Lyme disease spirochetes as compared with piroplasms may partially be explained by a differential human susceptibility to infection, but could also be influenced by the higher incidence of spirochetal infection in host-seeking nymphal ticks (Piesman et al. 1986~). Transmission efficiencies from infected ticks to hosts could also affect this risk, but previous studies (Donahue et al. 1987; Piesman and Spielman 1982), in addition to those reported here, confirm that one bite, from either a spirochete- or Babesia-infected nymph, is sufficient to infect P. leucopus. Transmission of both of these infectious agents is most efficient when ticks feed to repletion; spirochetes were rarely transmitted sooner than 24 hr following attachment of infected ticks (Piesman et al. 1987b), while Babesia sporozoites require a minimum of 36-48 hr to complete development in salivary glands (Piesman and Spielman 1980). Both infections can also be transmitted simultaneously by ticks concurrently infected with both pathogens (Piesman et al. 1987a). Interestingly, the prevalence of Lyme disease spirochetes in populations of hostseeking I. dammini appears to be double that of B. microti in locations where the prevalence of infection for either pathogen among rodent reservoirs is nearly the same (Piesman et al. 1986~; Spielman et al. 1981; Anderson et al. 1986). Although several factors could be responsible for this observation, we observed differences in spirochetal and babesial transmission efficiencies, from mouse reservoirs to vector ticks,
which could explain this twofold greater prevalence of Lyme spirochetes. It has been suggested that the apparently wider host range of B. burgdorferi-infected animals could serve to inoculate a larger proportion of larval I. dammini with this spirochete (Anderson and Magnarelli 1984). However, it has recently been demonstrated that mice (P. leucopus) alone may account for nearly 100% of the spirocheteinfected nymphal ticks, at least in Massachusetts, even in the presence of many of these other host animals (Mather et al. 1989b). Thus, some aspect of the efficiency with which these two pathogens infect vector ticks would appear to better explain the disparate pathogen prevalence rates in ticks. The intensity and duration of B. burgdorferi and B. microti infectivity from hamsters to ticks have recently been measured (Piesman 1988). In that study, a larger proportion of larval I. dammini became infected with Lyme spirochetes than with piroplasms after engorging on either of two animals simultaneously infected with both infectious agents. Although the infectivity to ticks for both pathogens was lower after 7 months than after 1 month postinfection, spirochetal infectivity was always greater than babesial infectivity. Using the natural rodent host for these infections, we found that B. burgdor-eri infected ticks more efficiently than did B. microti. In previous studies using P. leucopus, their duration of spirochetal infectivity was about equal to their duration of babesial infection (Donahue et al. 1987; Spielman et al. 1981), and this duration of infectivity (or infection) far exceeds the length of the natural 3- to 4month season for transmission of these two pathogens. Furthermore, where both agents are enzootic, mice are constantly inoculated during the spring and summer, presumably providing for continuous infection of these animals. Although the effect of continuous inoculation of either pathogen on the duration of infectivity has yet to be
INFECTION
EFFICIENCIES
OF TICK-BORNE
evaluated, natural populations of mice appear to be just as spirochete and Babesia infective for ticks feeding in May as they are in September (T. N. Mather, unpublished observations). An explanation for the disparate efficiency of transmission of B. burgdorferi and B. microti to ticks may partially be attributed to the intensity of infection in their natural mouse host. In mice, the intensity of B. microti infection is relatively low. Unlike in hamsters, where 50-60% of erythrocytes may become parasitized (Benach ef al. 1978; Piesman and Spielman 1982), the parasitemia rarely exceeds 0.1% of erythrocytes in naturally infected P. leucopus (Etkind et al. 1980; Spielman ef al. 1981). Because of this sparse parasitemia, ticks feeding on mice ingest fewer “gametocytes” (Rudzinska ef al. 1983b, 1984) which may, in turn, result in fewer successful tick infections than if the intensity of infection in mice was higher. Unfortunately, neither the intensity of an animal’s spirochetemia nor the precise mechanism of tick infection by B. burgdorferi has yet been assessed, thus it is impossible to compare the intensity of an animal’s spirochetal infection with its level of babesial infection. However, since the spirochetal infectivity to ticks reported here and in other studies (Donahue et al. 1987; Mather et al. 1989a, b; Piesman 1988) is consistently high, usually with greater than 75% of a feeding cohort becoming infected, it may be that spirochetes are relatively abundant in the tissue on which the tick feeds. The relative complexity in the mechanism of spirochetal and babesial infection of tick vectors may also explain their different transmission efficiencies. Piroplasms face several barriers before the tick becomes infective. In order to become infective (and be detected by the Feulgen stain), parasites must migrate from the tick’s gut to their salivary glands, undergoing syngamy and transforming from kinetes into sporoblasts (Piesman et al. 1986b). During
PATHOGENS
59
the course of tick infection, these parasites must traverse peritrophic membrane and gut epithelial cell barriers (Rudzinska et al. 1982, 1983a) in addition to undergoing several cellular transformations (Rudzinska et al. 1984). Lyme disease spirochetes, on the other hand, would appear to face fewer barriers in their transmission cycle. This bacterium sequesters in the gut of infected ticks; shortly after tick attachment to a host, spirochetes have been observed traversing the gut wall, in hemolymph, salivary glands (Zung et al. 1989), and eventually in the saliva (Benach et al. 1987; Ribeiro et al. 1987). Thus, ticks may be physiologically more permissive for spirochetal infection than for babesial infection, with the success of babesial infection in ticks being diminished by its more complex life cycle. We observed that the pathogen infection rates in ticks feeding on mice infected with both B. burgdorferi and B. microti appeared to be lower than those having fed on mice infected with a single agent. The observed statistical difference may be due to an inhibitory interaction at the level of the vertebrate host, one expressed within the tick, or both, yet no mechanism for this interaction is evident. Nevertheless, because most mice are concurrently infected (Anderson et al. 1986; unpublished results) such an inhibitory interaction may serve to diminish infection rates of both pathogens in naturally occurring ticks. Ticks become infected by B. burgdorferi with great efficiency, a fact which emphasizes the importance of finding interventions that interrupt this process to prevent Lyme disease. Schemes to prevent the successful feeding of immature ticks on P. leucopus (Mather et al. 1987), probably the most important reservoir for this infection (Mather et al. 1989b), have had the effect of greatly reducing spirochetal prevalence in some I. dummini populations (Mather et al. 1988). Because B. microti shares the same principal reservoir (Spielman et al. 1981;
60
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Anderson ef al. 1986, 19874 and is less prevalent in populations of nymphal I. dammini, such measures should prove even more effective in reducing transmission of this latter infection. ACKNOWLEDGMENTS This work was supported by a grant from The Arthritis Foundation. Additional support was provided by grants from the Chace Fund. David Arnold, and NIH (AI 1%93). REFERENCES ANDERSON,J. F.. DURAY, P. H., AND MAGNARELLI, L. A. 1987a. Prevalence of Borrelia burgdorferi in white-footed mice and fxodes dammini at Fort McCoy, Wis. Journal of Clinical Microbiology 25, 1495-1497. ANDERSON, MAGNARELLI,
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L. A. 1987b. Seasonal prevalence of Borrelia burgdorferi in natural populations of whitefooted mice, Peromyscus leucopus. Journal of Clinical Microbiology 25, 1X4-1566.
ANDERSON,
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L. A.. HYDE, F. W., AND MYERS, J. E. 1986. Peromyscus leucopus and Microtus pennsylvanicus simultaneously infected with Borrelia burgdorferi and Babesia microti. Journal of Clinical Microbiology
23, 135-137. ANDERSON, J. F., JOHNSON, L. A., HYDE, F. W., AND
R. C.,
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J. E. 1987~. Prevalence of Borrelia burgdorferi and Babesia microti in mice on islands inhabited by white-tailed deer. Applied and Environmental Microbiology 53, 892-894. ANDERSON, J. F., AND MAGNARELLI, L. A. 1984. Avian and mammalian hosts for spirochete-infected ticks and insects in a Lyme disease focus in Connecticut. Yale Journal of Biology and Medicine 57, 627-641. BENACH, J. L., COLEMAN, J. L., SKINNER, R. A., AND BOSLER. E. M. 1987. Adult Ixodes dammini on rabbits: A hypothesis for the development and transmission of Borrelia burgdorferi. Journal of Infecrious Diseases 155, 1300-1306. BENACH, J. L., WHITE, D. J., AND MCGOVERN, J. P. 1978. Babesiosis on Long Island: Host-parasite relationships of rodent and human derived Babesia microti isolates in hamsters. American Journal of Tropical Medicine and Hygiene 21, 1073-1078. BLEWETT, D. A., AND BRANAGAN, D., 1973. The demonstration of Theileria parva infection in intact Rhipicephalus appendiculatus salivary glands. MYERS,
Tropical Animal Health Products 5, 27-34.
G. J. 1986. Lyme disease: Its transmission and diagnostic features. Laboratory Management 24, 33-38. DAMMIN, G. J., SPIELMAN, A., BENACH, J., AND PIESMAN, J. 1981. The rising incidence of clinical Babesia microti infection. Human Pathology 12, 398-400. DONAHUE, J. G., PIESMAN, J., AND SPIELMAN, A. 1987. Reservoir competence of white-footed mice for Lyme disease spirochetes. American Journal of DAMMIN,
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P., PIESMAN, J., RUEBUSH, T. K., II, SPIELA., AND JLJRANEK, D. D. 1980. Methods for detecting Babesia microti infection in wild rodents. Journal of Parasitology 66, 107-l IO. MATHER, T. N., RIBEIRO, J. M. C., MOORE. S. I., AND SPIELMAN, A. 1988. Reducing transmission of the Lyme disease spirochete in a suburban setting. ETKIND, MAN,
Proceedings
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539, 402-403. MATHER, T. N., RIBEIRO, J. M. C., AND SPIELMAN, A. 1987. Lyme disease and babesiosis: Acaricide focused on potentially infected ticks. American Journal of Tropical Medicine
and Hygiene 36, 609-
614. T. N.. TELFORD, S. R., III, MACLACHLAN, A. B., AND SPIELMAN, A. 1989a. Incompetence of catbirds as reservoirs for the Lyme disease spirochete (Borrelia burgdorferi). Journal of Parasitology 75, 66-69. MATHER, T. N., WILSON, M. L., MOORE, S. I., RIBF.IRO, J. M. C., AND SPIELMAN, A. 1989b. Comparing the relative potential of rodents as reservoirs of the Lyme disease spirochete (Borrelia burgdorfen’). American Journal of Epidemiology 130, l43150. PIESMAN. J. 1988. Intensity and duration of Borrelia burgdorferi and Babesia microti infectivity in rodent Journal for Parasitology 18, hosts. International 687-689. PIESMAN, J., DONAHUE, J. G., MATHER, T. N., AND SPIELMAN, A. 1986a.Transovarially acquired Lyme disease spirochetes (Borrelia burgdorferi) in field collected larval Ixodes dammini (AcarhIxodidae).
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T. C., SINSKY, R. J., AND OBIRI, G. 1987a. Simultaneous transmission of Borrelia burgdorferi and Babesia microti by individual nymphal Ixodes dammini ticks. Journal of Clinical Microbiology 25, 2012-2013. PIESMAN, J., KARAKASHIAN, S. J., LEWENGRUB, S., RUDZINSKA, M. A., AND SPIELMAN, A. 1986b. Development of Babesia microti sporozoites in adult PIESMAN,
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Ixodes dammini. International tology 16, 381-385. PIESMAN, LEVINE,
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AND SPIELMAN, A. 1986~. Comparative prevalence of Babesia microti and Borrelia burgdorferi in four populations of lxodes dammini in eastern Massachusetts. Acta Tropica 43, 263-270. PIESMAN, J., MATHER, T. N., SINSKY, R. J., AND SPIELMAN, A. 1987b. Duration of tick attachment and Borrelia burgdorferi transmission. Journal of Clinical Microbiology 25, 557-558. PIESMAN, J., MATHER, T. N.. TELFORD, S. R., III, AND SPIELMAN. A. 1986d. Concurrent Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini. Journal of Clinical Microbiology 24, 44-7. PIESMAN, J., AND SPIELMAN, A. 1980. Human babesiosis on Nantucket Island: Prevalence of Babesia microti in ticks. American Journal of Tropical Medicine and Hygiene 29, 742-746. PIESMAN, J.. AND SPIELMAN. A. 1982. Babesia microti: Infectivity of parasites from ticks for hamsters and white-footed mice. Experimental Parasitology
53, 242-248. RIBEIRO, J. M. C., MATHER, T. N., PIESMAN. J.. AND SPIELMAN. A. 1987. Dissemination and salivary de-
livery of Lyme disease spirochetes in vector ticks (Acari:Ixodidae). Journal of Medical Entomology 24, 201-205. RUDZINSKA, M.. LEWENGRUB, S., SPIELMAN, A., AND PIESMAN, J. 1983a.Invasion of Babesia microti into epithelial cells of the tick gut. Journal of Proto:oology 30, 338-346. RUDZINSKA. M.. SPIELMAN. A., LEWENGRUB, S., PIESMAN, J., AND KARAKASHIAN. S. J. 1982. Pene-
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tration of the peritropic membrane of the tick by Babesia microti. Cell and Tissue Research 221,471-
481. RUDZINSKA, M.. SPIELMAN. A., LEWENGRUB, S.. PIESMAN, J., AND KARAKASHIAN, S. J. 1984. The sequence of developmental events of Babesia micron in the gut of lxodes dammini. Protistologica
20, 649-663. RUDZINSKA. M., SPIELMAN, A., LEWENCRUB. S., TRAGER, W., AND PIESMAN, J. 1983b. Sexuality in
piroplasms as revealed by electron microscopy in Babesia microti. Proceedings of the National Academy of Sciences USA 80, 2966-2970. SPIELMAN, A., ETKIND. P.. PIESMAN. J., RUEBUSH, T. K., II, JURANEK, D. D., AND JACOBS, M. S.
1981. Reservoir hosts of human babesiosis on Nantucket Island. American Journal of Tropical Medicine and Hygiene 30, 560-565. STEERE, A. C.. GRODZICKI, R. L., KORNBLATT, A. N., CRAFT, 3. E.. BARBOUR, A. G., BURGDORFER. W.. SCHMID, G. P., JOHNSON, E., AND MALAWISTA. S. F. 1983.The spirochetal etiology of Lyme disease. Netc England Journal of Medicine
308, 734-739. ZUNG, J. L.. LEWENGRUB, S., RUDZINSKA, M. A., SPIELMAN, A., TELFORD, S. R.. III, AND PIESMAN,
J. 1989. Fine structural evidence for the penetration of the Lyme disease spirochete Borrelia burgdorferi through the gut and salivary tissues of Ixodes dammini. Canadian Journal of Zoology, 61, 1737-1748. Received 4 April 1989: accepted with revision 21 August 1989.
Borrelia
burgdorferi and Babe& microti: Efficiency of Transmiss’ion from Reservoirs to Vector Ticks (/iodes dammini) THOMASN.MATHER,'SAM R. TELFORD III, SEAN I. MOORE, AND ANDREW SPIELMAN
Department
of Tropical Public Health. Harvard School of Public Health, Bosion, Massachusetts 02115, U.S.A.
665 Huntington
Avenue,
MATHER, T. N., TELFORD, S. R., III, MOORE,S. I., AND SPIELMAN, A. 1990. Borrelia burgdorferi and Babesia microti: Efftciency of transmission from reservoirs to vector ticks @odes dammini). Experimental Parasitology 70, 55-61. In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically. spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas. e 1990 Academic PESS, IIIC. INDEX DESCRIPTORS: Lyme disease, Borrelia burgdorferi; Babesiosis, Babesia mirroti; Ixodes dammini; Transmission: Peromyscus leucopus.
croti throughout the season of activity of the nymphal tick. The result is nearly universal infection of the reservoir. Estimates of spirochetal prevalence in mice evaluated during several field surveys have demonstrated that about 90% were infected with spirochetes (Anderson et al. 1987a, b; Mather et al. 1989a, b), while Babesia was detected in nearly 80% of the animals sampled during late summer (Etkind et a/. 1980; Spielman et a/. 1981). Therefore, since larval I. dammini must acquire these infectious agents during bloodfeeding, due to the relative lack of inherited infection (Piesman et al. 1986a), their likelihood of encountering a mouse infective for either pathogen would appear to be similar. Paradoxically, despite a high degree of prevalence for both B. burgdorferi and B. microti in reservoir populations, the rate of infection in field-derived vector ticks for these two infectious agents differs.
In the northeastern United States, the white-footed mouse (Peromyscus leucopus) is the. principal reservoir for both the Lyme disease spirochete, Borrelia burgdorferi, and the piroplasm, Babesia microti (Mather et al. 1989b; Spielman et al. 1981). This animal also serves as a major host for immature txodes dammini, the nymphal stage of which is the main tick vector of both infectious agents. Indeed, in nature, nymphal ticks may be concurrently infected with both pathogens (Piesman et al. 1986d), and may transmit both infections simultaneously (Piesman et a/. 1987a). In areas where transmission is intense, mice, as well as other animals, are thus continually inoculated with both B. burgdorferi and B. mi’ Present address: Department of Population Sciences, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 021IS. 55
0014-4894190$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
MATHER ET AL.
Nymphal 1. dammini collected from sites where both pathogens occur in the reservoir have nearly twice the rate of infection for spirochetes as for Babesia (Piesman et al. 1986~). Perhaps some aspect of the reservoir-vector-pathogen dynamics favors spirochetal infection of ticks over their infection by Babesia. Relative efficiencies of pathogen acquisition by ticks, in addition to the success of transstadial passage of their infection, may favor spirochetal infection. Accordingly, we determined the efficiency with which I. dammini, feeding on mice, were infected with either pathogen in laboratory experiments. We measured the prevalence of infection in nymphal ticks previously fed as larvae on these spirochete- or B. microti-infected mice, as well as on mice infected by both pathogens concurrently. MATERIALS
AND METHODS
White-footed mice were from a colony originally derived from several Massachusetts locations, but have been maintained in the laboratory for more than four generations. These mice are free of any natural 8. burgdorferi or 8. microti infection, as determined by tick xenodiagnosis, and examination of blood smears. Mice used for each replicate of the experiment were. in every case but one, age- and sex-matched, and in all but two cases were litter mates. To begin each experiment, mice were infected with B.‘burgdorferi, B. microti, or with both pathogens simultaneously. Animals were infected by allowing either three nymphal I. dammini from a spirocheteinfected cohort or six nymphs from a piroplasminfected cohort to feed to repletion on the mice. To produce concomitant infections, three nymphs from the spirochete-infected cohort were fed on mice simultaneously with six nymphs from the piroplasminfected cohort. The numbers of infecting nymphs used reflected the difference in the cohort infection rate for either pathogen and ensured that each mouse received at least one infectious bite. “Infecting” nymphs were produced by allowing larvae to acquire spirochetes from a mouse previously infected by tick inoculation of the JDI strain of B. burgdorferi (Piesman et al. 1987a)or Babesia from a hamster infected with the GI strain of B. microri (Piesman and Spielman 1982) which has been maintained by alternating tick and syringe passages through golden hamsters. After feeding on the experimental mice, replete infecting nymphs were collected as they detached and were ex-
amined for evidence of infection by either spirochetes or piroplasms, as described below. Three weeks following the initial tick feeding, about 200 larval I. dammini, from egg batches determined to be free of inherited spirochetal infection, were allowed to infest the experimentally infected mice. These larvae were the offspring of field-swept females fed on New Zealand white rabbits in the laboratory. Samples (25-50) of unfed larvae from each egg batch used were examined, without knowledge of the sample’s origin, by a direct fluorescent antibody assay (described below). Larvae engorging on the experimentally infected mice were collected as they detached, were placed in plastic vials and stored in humid chambers at about 95% RH, and were held at 22°C and a l6-hr photophase until nymphs emerged. To determine the prevalence of infection in ticks, nymphs were examined 34 weeks after molting. To detect spirochetes. tick midgut tissues were dissected into a drop of phosphate-buffered saline (pH 7.3) on a glass microscope slide; this tissue was smeared onto the slide with a cover glass, air dried, and fixed in an acetone bath before storage at -20°C. Later, slides were treated with a I:100 dilution of a fluorescein isothiocyanate-conjugated polyclonal antibody prepared from two New Zealand white rabbits inoculated with the Guilford strain of B. burgdorferi (Steere ef al. 1983)and then examined at 400x magnification by fluorescent microscopy. To detect babesial sporozoites, tick salivary glands were similarly dissected from ticks that were prefed on noninfected Syrian golden hamsters for 54 hr (Piesman and Spielman 1982). Glands were allowed to dry onto a gelatin-coated microscope slide, fixed with methanol before storage, and later stained by a modified Feulgens reaction before examination (Piesman et al. 1986~; Blewett and Branagan 1973). In addition, experimentally infected mice were examined for the presence of intraerythrocytic B. microfi infection both before and after tick feeding. Thin blood smears were made from tail snips, dried, fixed in absolute methanol, and stained with IO%,Giemsa at pH 7.0. All slides were examined at 400x magnification for a total of 100 random fields.
RESULTS
We determined whether experimental mice were inoculated with B. burgdorferi, B. microti, or with both infectious agents by examining infecting ticks upon their detachment. In every case, at least one fully engorged nymph, infected with the appropriate agent, was recovered (Table I). Additionally, mice bitten by B. microtiinfecting nymphs all became infected
INFECTION
EFFICIENCIES
OF TICK-BORNE
57
PATHOGENS
rochetes and Bubesiu exhibited infection rates of 82 and 34%, respectively. The spirochetal infection rate was always greater than 75% for those cohorts of ticks feeding on spirochete only infected mice, and was Frequency of usually similar but always greater than 50% infection in for those cohorts feeding on dually infected “infecting” nymphs animals (Table II). These slight differences recovered Kind of Number in infection rates among concurrently inof mice 1 2 3 4 5 6 infection fected nymphs, when compared to nymphs Spirochete IO 433--infected with just one of the pathogens, Piroplasm IO 5 3 1 I 0 0 may not be explained by random variation Concurrent 9 (Fisher’s exact test: spirochete, z, = -3.03, Spirochete 324--P < 0.05; piroplasm, z, = -2.26, P < Piroplasm 2 2 3 2 0 0 0.05). The babesial infection rate among the tick cohorts varied, depending on the aniwithin the 3-week incubation period, as mal tested, although more than two-thirds demonstrated by positive blood smears. No of the cohorts exhibited rates lower than corresponding test exists to detect spiro- 50%. However, one Bubesiu-only infected chetal infection in animals; however, Don- mouse did infect 12 of 15 ticks examined. ahue et al. (1987) demonstrated that mice To assess whether the frequency of inocbecame infective as early as 1 week postin- ulation affected an animal’s infectivity to fectious tick bite. ticks, we compared the number of infected We also determined the prevalence of in- infecting nymphal ticks recovered (Table I) fection in nymphal ticks which had fed as with the pathogen infection rate of tick larvae on these spirochete-, Bubesia-, or cohorts from individual mice (Table II). concurrently infected mice. An average of Correlation coefficients determined for spi92% of nymphs, derived from larvae en- rochete- and piroplasm-infected mice (R’ = gorged on B. burgdor-eri-infected mice, 0.1559; P = 0.667 and R2 = 0.1997; P = proved infected (Table II). In contrast, 0.58, respectively) indicated that the freBabesia sporozoites were detected in only quency of inoculation, within the range of 1 45% of those nymphs derived from larvae to 3 (or 4 in the case of B. microti-infecting feeding on the Bubesiu-infected mice. Ticks nymphs) did not affect an animal’s infectivfrom mice with concurrent infections of spi- ity to ticks. Similar results were obtained TABLE I Frequency of Inoculation of Mice (P. leucopus) Bitten by Nymphal Ticks (I. dammini) Infected with Lyme Spirochetes (B. burgdorferi), Piroplasms (B. microtn, or Both Pathogens
TABLE II Efficiency of Pathogen Acquisition by Ticks (I. dammini) Engorging on Mice (P. leucopus) Infected with Lyme Disease Spirochetes (B. burgdorferi), Piroplasms (B. microti). or with Both Pathogens
Kind of infection Spirochete Piroplasm
Concurrent Spirochete Piroplasm
Number of mice IO IO 9
Distribution of tick cohorts by percent infected
Derived nymphs Number
% infected
~25%
26-50%
.51-75%
215
92% 45%
0 3
0 3
0 3
IO
176 190 159
82% 34%
0 5
0 2
3 2
6 0
~76%
1
58
MATHERETAL.
for mice concurrently infected with spirochetes and piroplasms (R2 = 0.1571; P = 0.687 and R2 =0.2895; P = 0.45, respectively). DISCUSSION
Human cases of Lyme disease are more prevalent than are cases of babesiosis (Dammin ef al. 1981; Dammin 1986). Where both pathogens occur together, the greater risk of acquiring infection associated with Lyme disease spirochetes as compared with piroplasms may partially be explained by a differential human susceptibility to infection, but could also be influenced by the higher incidence of spirochetal infection in host-seeking nymphal ticks (Piesman et al. 1986~). Transmission efficiencies from infected ticks to hosts could also affect this risk, but previous studies (Donahue et al. 1987; Piesman and Spielman 1982), in addition to those reported here, confirm that one bite, from either a spirochete- or Babesia-infected nymph, is sufficient to infect P. leucopus. Transmission of both of these infectious agents is most efficient when ticks feed to repletion; spirochetes were rarely transmitted sooner than 24 hr following attachment of infected ticks (Piesman et al. 1987b), while Babesia sporozoites require a minimum of 36-48 hr to complete development in salivary glands (Piesman and Spielman 1980). Both infections can also be transmitted simultaneously by ticks concurrently infected with both pathogens (Piesman et al. 1987a). Interestingly, the prevalence of Lyme disease spirochetes in populations of hostseeking I. dammini appears to be double that of B. microti in locations where the prevalence of infection for either pathogen among rodent reservoirs is nearly the same (Piesman et al. 1986~; Spielman et al. 1981; Anderson et al. 1986). Although several factors could be responsible for this observation, we observed differences in spirochetal and babesial transmission efficiencies, from mouse reservoirs to vector ticks,
which could explain this twofold greater prevalence of Lyme spirochetes. It has been suggested that the apparently wider host range of B. burgdorferi-infected animals could serve to inoculate a larger proportion of larval I. dammini with this spirochete (Anderson and Magnarelli 1984). However, it has recently been demonstrated that mice (P. leucopus) alone may account for nearly 100% of the spirocheteinfected nymphal ticks, at least in Massachusetts, even in the presence of many of these other host animals (Mather et al. 1989b). Thus, some aspect of the efficiency with which these two pathogens infect vector ticks would appear to better explain the disparate pathogen prevalence rates in ticks. The intensity and duration of B. burgdorferi and B. microti infectivity from hamsters to ticks have recently been measured (Piesman 1988). In that study, a larger proportion of larval I. dammini became infected with Lyme spirochetes than with piroplasms after engorging on either of two animals simultaneously infected with both infectious agents. Although the infectivity to ticks for both pathogens was lower after 7 months than after 1 month postinfection, spirochetal infectivity was always greater than babesial infectivity. Using the natural rodent host for these infections, we found that B. burgdor-eri infected ticks more efficiently than did B. microti. In previous studies using P. leucopus, their duration of spirochetal infectivity was about equal to their duration of babesial infection (Donahue et al. 1987; Spielman et al. 1981), and this duration of infectivity (or infection) far exceeds the length of the natural 3- to 4month season for transmission of these two pathogens. Furthermore, where both agents are enzootic, mice are constantly inoculated during the spring and summer, presumably providing for continuous infection of these animals. Although the effect of continuous inoculation of either pathogen on the duration of infectivity has yet to be
INFECTION
EFFICIENCIES
OF TICK-BORNE
evaluated, natural populations of mice appear to be just as spirochete and Babesia infective for ticks feeding in May as they are in September (T. N. Mather, unpublished observations). An explanation for the disparate efficiency of transmission of B. burgdorferi and B. microti to ticks may partially be attributed to the intensity of infection in their natural mouse host. In mice, the intensity of B. microti infection is relatively low. Unlike in hamsters, where 50-60% of erythrocytes may become parasitized (Benach ef al. 1978; Piesman and Spielman 1982), the parasitemia rarely exceeds 0.1% of erythrocytes in naturally infected P. leucopus (Etkind et al. 1980; Spielman ef al. 1981). Because of this sparse parasitemia, ticks feeding on mice ingest fewer “gametocytes” (Rudzinska ef al. 1983b, 1984) which may, in turn, result in fewer successful tick infections than if the intensity of infection in mice was higher. Unfortunately, neither the intensity of an animal’s spirochetemia nor the precise mechanism of tick infection by B. burgdorferi has yet been assessed, thus it is impossible to compare the intensity of an animal’s spirochetal infection with its level of babesial infection. However, since the spirochetal infectivity to ticks reported here and in other studies (Donahue et al. 1987; Mather et al. 1989a, b; Piesman 1988) is consistently high, usually with greater than 75% of a feeding cohort becoming infected, it may be that spirochetes are relatively abundant in the tissue on which the tick feeds. The relative complexity in the mechanism of spirochetal and babesial infection of tick vectors may also explain their different transmission efficiencies. Piroplasms face several barriers before the tick becomes infective. In order to become infective (and be detected by the Feulgen stain), parasites must migrate from the tick’s gut to their salivary glands, undergoing syngamy and transforming from kinetes into sporoblasts (Piesman et al. 1986b). During
PATHOGENS
59
the course of tick infection, these parasites must traverse peritrophic membrane and gut epithelial cell barriers (Rudzinska et al. 1982, 1983a) in addition to undergoing several cellular transformations (Rudzinska et al. 1984). Lyme disease spirochetes, on the other hand, would appear to face fewer barriers in their transmission cycle. This bacterium sequesters in the gut of infected ticks; shortly after tick attachment to a host, spirochetes have been observed traversing the gut wall, in hemolymph, salivary glands (Zung et al. 1989), and eventually in the saliva (Benach et al. 1987; Ribeiro et al. 1987). Thus, ticks may be physiologically more permissive for spirochetal infection than for babesial infection, with the success of babesial infection in ticks being diminished by its more complex life cycle. We observed that the pathogen infection rates in ticks feeding on mice infected with both B. burgdorferi and B. microti appeared to be lower than those having fed on mice infected with a single agent. The observed statistical difference may be due to an inhibitory interaction at the level of the vertebrate host, one expressed within the tick, or both, yet no mechanism for this interaction is evident. Nevertheless, because most mice are concurrently infected (Anderson et al. 1986; unpublished results) such an inhibitory interaction may serve to diminish infection rates of both pathogens in naturally occurring ticks. Ticks become infected by B. burgdorferi with great efficiency, a fact which emphasizes the importance of finding interventions that interrupt this process to prevent Lyme disease. Schemes to prevent the successful feeding of immature ticks on P. leucopus (Mather et al. 1987), probably the most important reservoir for this infection (Mather et al. 1989b), have had the effect of greatly reducing spirochetal prevalence in some I. dummini populations (Mather et al. 1988). Because B. microti shares the same principal reservoir (Spielman et al. 1981;
60
MATHER ET AL.
Anderson ef al. 1986, 19874 and is less prevalent in populations of nymphal I. dammini, such measures should prove even more effective in reducing transmission of this latter infection. ACKNOWLEDGMENTS This work was supported by a grant from The Arthritis Foundation. Additional support was provided by grants from the Chace Fund. David Arnold, and NIH (AI 1%93). REFERENCES ANDERSON,J. F.. DURAY, P. H., AND MAGNARELLI, L. A. 1987a. Prevalence of Borrelia burgdorferi in white-footed mice and fxodes dammini at Fort McCoy, Wis. Journal of Clinical Microbiology 25, 1495-1497. ANDERSON, MAGNARELLI,
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614. T. N.. TELFORD, S. R., III, MACLACHLAN, A. B., AND SPIELMAN, A. 1989a. Incompetence of catbirds as reservoirs for the Lyme disease spirochete (Borrelia burgdorferi). Journal of Parasitology 75, 66-69. MATHER, T. N., WILSON, M. L., MOORE, S. I., RIBF.IRO, J. M. C., AND SPIELMAN, A. 1989b. Comparing the relative potential of rodents as reservoirs of the Lyme disease spirochete (Borrelia burgdorfen’). American Journal of Epidemiology 130, l43150. PIESMAN. J. 1988. Intensity and duration of Borrelia burgdorferi and Babesia microti infectivity in rodent Journal for Parasitology 18, hosts. International 687-689. PIESMAN, J., DONAHUE, J. G., MATHER, T. N., AND SPIELMAN, A. 1986a.Transovarially acquired Lyme disease spirochetes (Borrelia burgdorferi) in field collected larval Ixodes dammini (AcarhIxodidae).
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T. C., SINSKY, R. J., AND OBIRI, G. 1987a. Simultaneous transmission of Borrelia burgdorferi and Babesia microti by individual nymphal Ixodes dammini ticks. Journal of Clinical Microbiology 25, 2012-2013. PIESMAN, J., KARAKASHIAN, S. J., LEWENGRUB, S., RUDZINSKA, M. A., AND SPIELMAN, A. 1986b. Development of Babesia microti sporozoites in adult PIESMAN,
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AND SPIELMAN, A. 1986~. Comparative prevalence of Babesia microti and Borrelia burgdorferi in four populations of lxodes dammini in eastern Massachusetts. Acta Tropica 43, 263-270. PIESMAN, J., MATHER, T. N., SINSKY, R. J., AND SPIELMAN, A. 1987b. Duration of tick attachment and Borrelia burgdorferi transmission. Journal of Clinical Microbiology 25, 557-558. PIESMAN, J., MATHER, T. N.. TELFORD, S. R., III, AND SPIELMAN. A. 1986d. Concurrent Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini. Journal of Clinical Microbiology 24, 44-7. PIESMAN, J., AND SPIELMAN, A. 1980. Human babesiosis on Nantucket Island: Prevalence of Babesia microti in ticks. American Journal of Tropical Medicine and Hygiene 29, 742-746. PIESMAN, J.. AND SPIELMAN. A. 1982. Babesia microti: Infectivity of parasites from ticks for hamsters and white-footed mice. Experimental Parasitology
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livery of Lyme disease spirochetes in vector ticks (Acari:Ixodidae). Journal of Medical Entomology 24, 201-205. RUDZINSKA, M.. LEWENGRUB, S., SPIELMAN, A., AND PIESMAN, J. 1983a.Invasion of Babesia microti into epithelial cells of the tick gut. Journal of Proto:oology 30, 338-346. RUDZINSKA. M.. SPIELMAN. A., LEWENGRUB, S., PIESMAN, J., AND KARAKASHIAN. S. J. 1982. Pene-
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481. RUDZINSKA, M.. SPIELMAN. A., LEWENGRUB, S.. PIESMAN, J., AND KARAKASHIAN, S. J. 1984. The sequence of developmental events of Babesia micron in the gut of lxodes dammini. Protistologica
20, 649-663. RUDZINSKA. M., SPIELMAN, A., LEWENCRUB. S., TRAGER, W., AND PIESMAN, J. 1983b. Sexuality in
piroplasms as revealed by electron microscopy in Babesia microti. Proceedings of the National Academy of Sciences USA 80, 2966-2970. SPIELMAN, A., ETKIND. P.. PIESMAN. J., RUEBUSH, T. K., II, JURANEK, D. D., AND JACOBS, M. S.
1981. Reservoir hosts of human babesiosis on Nantucket Island. American Journal of Tropical Medicine and Hygiene 30, 560-565. STEERE, A. C.. GRODZICKI, R. L., KORNBLATT, A. N., CRAFT, 3. E.. BARBOUR, A. G., BURGDORFER. W.. SCHMID, G. P., JOHNSON, E., AND MALAWISTA. S. F. 1983.The spirochetal etiology of Lyme disease. Netc England Journal of Medicine
308, 734-739. ZUNG, J. L.. LEWENGRUB, S., RUDZINSKA, M. A., SPIELMAN, A., TELFORD, S. R.. III, AND PIESMAN,
J. 1989. Fine structural evidence for the penetration of the Lyme disease spirochete Borrelia burgdorferi through the gut and salivary tissues of Ixodes dammini. Canadian Journal of Zoology, 61, 1737-1748. Received 4 April 1989: accepted with revision 21 August 1989.
