Abstract
The sensitivity of Borrelia burgdorferi sensu lato to animal sera was analyzed. Complement-mediated borreliacidal effects were observed with particular combinations of host serum and Borrelia genospecies. The species-specific pattern of viability and/or lysis is highly consistent with the pattern of reservoir competence of hosts for B. burgdorferi sensu lato, suggesting a key role of complement in the global ecology of Lyme borreliosis.
Borrelia burgdorferi, the tick-borne agent of Lyme borreliosis, is a genetically diverse species complex of spirochetes, comprising at least nine genospecies (22, 23, 31). The different genospecies of B. burgdorferi sensu lato appear to cause distinct clinical manifestations of Lyme borreliosis in humans (28). Consistent with this, arthritis is common in North America, where B. burgdorferi sensu stricto predominates, whereas in Eurasia, neuroborreliosis and acrodermatitis are typical disease manifestations, reflecting the high abundance of Borrelia garinii and Borrelia afzelii throughout this region (5, 7, 16, 20, 25). Other genospecies, such as Borrelia lusitaniae (former genomic group PotiB2), Borrelia valaisiana (former genomic group VS116), and Borrelia japonica from Japan or Borrelia andersonii from the United States, have not yet been associated with disease in humans. Geographic variation in the distribution of different B. burgdorferi sensu lato genospecies in regions where the same tick species is involved as the vector, e.g., in Europe with Ixodes ricinus (10, 23–25), appears to be associated with host-specific differential spirochete transmission between hosts and ticks. For example, recent studies indicate that B. garinii and B. valaisiana are transmitted preferentially to ticks by certain avian hosts while B. afzelii is transmitted preferentially to ticks by European rodent species (6, 7, 14, 19). Although it has been previously suggested that adaptive immune responses may be involved in the regulation of spirochete transmission (11), the mechanisms underlying differential transmission of the Borrelia genospecies by hosts have so far been unknown.
Cultured spirochetes belonging to different genospecies of B. burgdorferi sensu lato differ in their sensitivity to human serum (1, 29). To determine whether such a phenomenon plays a role in spirochete transmission cycles, we analyzed the sensitivity of different genospecies of B. burgdorferi sensu lato to sera from a wide range of reservoir-competent and -incompetent animals, including small mammals, lagomorphs, sheep, horses, cattle, pigs, deer, and game birds.
The Borrelia strains ZS 7 (B. burgdorferi sensu stricto), ACA-1 (B. afzelii), ZQ 1 (B. garinii), HO 14 (B. japonica), and UK (B. valaisiana) were cultured in BSK-II medium (Sigma) without the addition of serum or antibiotics. The isolates were low passage and clonal. The taxonomic identity and clonality of each isolate were confirmed by PCR amplification of the intergenic spacer between the 5S and 23S rRNA genes and subsequent genotyping by using the reverse line blot as described previously (14, 23).
Sera were taken from adult wood mice (Apodemus sylvaticus), BALB/c mice, bank voles (Clethrionomys glareolus), Syrian hamsters, grey squirrels (Sciurus carolinensis), blue mountain hares (Lepus timidus), sheep (lambs), horses, pigs, cattle (cow and calf), red deer (Cervus elaphus), pheasants (Phasianus colchicus), and a human. All sera, except those from the human, hares, and horses, came from animals without any previous contact with I. ricinus ticks. The following noninfected animals were bred at the institutions indicated: small rodents at the Department of Zoology, University of Oxford, and NERC Institute of Virology and Environmental Microbiology, Oxford, United Kingdom; pheasants at the Game Conservancy Trust, Fordingbridge, United Kingdom; sheep (lambs), pigs, and cattle at the Department of Veterinary Clinical Medicine, University of Liverpool, Liverpool, United Kingdom; and deer at the Reediehill Deer Farm, Auchtermuchty, United Kingdom.
For borreliacidal assays, Borrelia cultures were grown to an approximate density of 107 cells per ml of culture medium at 33°C without antibiotics under microaerophilic conditions. Sera were thawed on ice shortly before use. Suspensions of Borrelia cultures were added to variable concentrations of serum to give final volumes of Borrelia-serum suspensions of 100 μl per well in 96-well microtiter plates (Nunc). Some aliquots of sera were depleted of functional complement, or components thereof, by heat treatment (0.5 h of incubation at 56°C) or by the addition of either EDTA (10 mM) or MgCl2 (4 mM) plus EGTA (10 mM), while other aliquots were left untreated. Controls consisted of Borrelia cultures without any supplements. The plates were sealed and incubated at 33°C for mammalian sera and at either 33 or 38°C for avian sera. Three parameters of borreliacidal effects, each known to be an indicator of Borrelia mortality (29), were recorded: bleb formation, immobilization, and bacteriolysis. Each well was read at 0, 2, 4, and 22 h. Ten microliters of each suspension was collected aseptically and examined by using dark-field microscopy.
After incubation for 22 h with a 50% final serum concentration, a clear pattern of bacteriolysis was observed (Table 1). All rodent and human sera were borreliacidal for B. garinii and B. valaisiana but not for B. afzelii and B. japonica. In contrast, all pheasant sera showed the reverse pattern, killing B. afzelii and B. japonica but not B. garinii and B. valaisiana. Sera from wood mice and pheasants were also partially borreliacidal for B. burgdorferi sensu stricto. The hare serum was borreliacidal for the genospecies tested except for B. japonica. Most sheep sera were intermediately borreliacidal for B. burgdorferi sensu stricto, B. afzelii, and B. japonica but highly borreliacidal for B. garinii and B. valaisiana. The sera from horses, pigs, and cattle killed all of the genospecies tested except for B. burgdorferi sensu stricto, while the sera from red deer were indiscriminately borreliacidal for all of the genospecies tested. The pattern of Borrelia sensitivity to the human serum included in the present study was consistent with that reported in earlier studies (1, 29). Controls, including all of the Borrelia strains, which were always incubated together with the test samples at either 33 or 38°C, showed no genotype-specific difference in temperature tolerance per se (data not shown). Furthermore, incubation of bird serum and spirochetes at 33°C showed the same pattern of results as that observed at 38°C (data not shown). Thus, although the body temperature of birds is higher than that of mammals, differences in the temperature tolerance of spirochete strains cannot account for the observed contrasting patterns of bacteriolytic activity of sera from birds and rodents.
TABLE 1.
Bacteriolysis of different genospecies of B. burgdorferi sensu lato by untreated sera after 22 h of incubation
| Species | % Bacteriolysisa
|
|||||
|---|---|---|---|---|---|---|
| Serum sample | B. burgdorferi | B. garinii | B. valaisiana | B. afzelii | B. japonica | |
| Wood mouse | 1 | 29 | + | + | − | − |
| 2 | 16 | + | NT | − | − | |
| 3 | 31 | + | NT | − | − | |
| Mouse (BALB/c) | 1 | 17 | + | + | − | − |
| Vole | 1 | − | + | + | − | − |
| 2 | − | + | + | − | − | |
| 3 | − | + | + | − | − | |
| Hamster | 1 | − | + | + | − | − |
| 2 | − | + | + | − | − | |
| Squirrel | 1 | − | + | + | − | − |
| Human | 1 | − | + | + | − | − |
| Pheasant | 1 | 32 | − | − | + | + |
| 2 | 68 | − | − | + | + | |
| 3 | 20 | − | − | + | + | |
| Hare | 1 | + | + | + | + | − |
| Sheep | 1 | 8 | + | NR | 22 | + |
| 2 | 45 | + | + | 26 | 70 | |
| 3 | 38 | + | + | 35 | 62 | |
| 4 | 84 | + | + | NR | 27 | |
| Horse | 1 | 21 | + | + | + | + |
| 2 | 15 | + | + | + | + | |
| 3 | 26 | + | + | + | + | |
| 4 | 25 | + | + | + | + | |
| Pig (pool) | 36 | + | + | + | + | |
| Bovine calf (pool) | − | + | + | + | + | |
| Cow (pool) | 92 | + | + | + | + | |
| Deer | 1 | + | + | + | + | + |
| 2 | + | + | + | + | + | |
| 3 | + | + | + | + | + | |
| 4 | + | + | + | + | + | |
Borreliacidal effects were scored as >95% (+) and <5% (−) mortality of spirochetes. Number values are given for intermediate effects. NT, not tested; NR, not readable.
Borreliacidal effects became apparent within 2 h of incubation and increased with time. For the selected combinations of wood mouse-B. garinii, sheep-B. burgdorferi sensu stricto, red deer-B. garinii, and pheasant-B. afzelii, the effect of the serum concentration on the kinetics of killing was assayed (Table 2). Partial effects were observed at a serum concentration of 10%, but all effects were more rapid and pronounced at the higher concentrations of 25 and 50%.
TABLE 2.
Bacteriolysis of B. burgdorferi sensu lato by different concentrations of untreated sera after 2, 4, and 22 h of incubationa
| Animal speciesspirochete | Serum concn (%) | % Bacteriolysis at incubation time (h):
|
||
|---|---|---|---|---|
| 2 | 4 | 22 | ||
| Mouse-B. garinii | 10 | 5 | 24 | 61 |
| 25 | 42 | 100 | 100 | |
| 50 | >95 | 100 | 100 | |
| Pheasant-B. afzelii | 10 | 0 | 11 | 31 |
| 25 | 11 | 19 | 87 | |
| 50 | 17 | 28 | 100 | |
| Sheep-B. burgdorferi | 10 | 5 | 13 | 21 |
| 25 | 9 | 12 | 43 | |
| 50 | 14 | 18 | 84 | |
| Deer-B. garinii | 10 | 72 | 78 | 100 |
| 25 | 100 | 100 | 100 | |
| 50 | 100 | 100 | 100 | |
For each combination of animal species and spirochetal strain, the percent mortality of spirochetes is given for one representative untreated serum sample.
Heat inactivation (0.5 h at 56°C) of sera from mice (n = 3), hares (n = 1), sheep (n = 4), cows (pool), bovine calves (pool), pigs (pool), deer (n = 4), and pheasants (n = 3) abolished all borreliacidal effects, indicating that the complement system is involved in the killing. When these sera were supplemented with EDTA, all borreliacidal effects were abolished in the same way as seen after heat inactivation, whereas supplementation with Mg-EGTA did not disrupt bacteriolysis (data not shown). These results indicate that the alternative pathway of the complement system, which operates independently of antibodies, plays a key role in spirochete killing (30). Thus, the bacteriolysis observed in the present study is distinct from the borreliacidal effects observed with immune sera (12, 21).
All genospecies of B. burgdorferi sensu lato are maintained in nature by tick-vertebrate transmission cycles. The pattern of species-specific, complement-mediated sensitivity to serum observed in the present study is highly consistent with the observations on spirochete transmission patterns reported so far. For example, the resistance of B. afzelii to rodent serum parallels the high level of transmission competence of both small natural rodent species (7) and squirrels (2) for this genospecies. In contrast, the particular B. garinii strain used in the present study, isolated in western Europe, was readily lysed by rodent sera. This is consistent with the finding that European rodents are inefficient reservoir hosts for the variants of B. garinii found in Europe, despite the fact that B. garinii has been isolated from or detected in rodent tissues (7, 14, 16, 17). Most interestingly in terms of evolution, B. garinii is a polymorphic genospecies of B. burgdorferi sensu lato (31), and there is evidence that one genotype of this genospecies, detected only in Japan so far, is efficiently transmitted by Japanese rodents to ticks (16). The resistance of the B. garinii strain tested against bird serum, on the other hand, is consistent with the finding that B. garinii has been associated with ticks derived from seabirds (19), blackbirds (6), and pheasants (13, 14). Likewise, blackbirds and pheasants, but not rodents, have been identified as reservoir hosts for B. valaisiana (6, 14). The finding that pheasant serum lysed B. afzelii, by comparison, may explain the incompetence of such birds as reservoirs for this genospecies. Sera from wood mice, but not from bank voles, were intermediately borreliacidal for B. burgdorferi sensu stricto, which is consistent with the lower degree of reservoir competence of wood mice for this genospecies compared with that of bank voles (11). Furthermore, the reported reservoir incompetence of deer (8, 27) correlates with the indiscriminatory borreliacidal activity of deer sera against all the genospecies tested. The reservoir competence for B. burgdorferi sensu lato of another ungulate species, sheep, appears to be low. However, it has been shown that transmission of B. burgdorferi sensu stricto from infected to uninfected ticks cofeeding on sheep may occur (18), perhaps related to the intermediate sensitivity of this genospecies to sheep serum.
In summary, the pattern of serum (complement) sensitivity of different Borrelia genospecies matches the known reservoir status of many vertebrate species for B. burgdorferi sensu lato. The hare, however, appears to be an exception since this species has been described to be reservoir competent for B. burgdorferi sensu lato in Scandinavia (26). The reason for the discrepancy between the pattern of serum compatibility and the reported reservoir status of hares remains unclear but may be related to the particular source of the serum (a hare from Scotland) and the strains of B. burgdorferi sensu lato used in the present study. At present, there is no information available on the reservoir competence of horses, pigs, and cattle for B. burgdorferi sensu lato. However, by extrapolating the findings of the present study, one may predict that horses, pigs (perhaps also wild boar), and cattle are reservoir competent, at a low level, for B. burgdorferi sensu stricto but not for any other genospecies analyzed in the present study.
Vertebrate hosts may be infected concurrently with different genospecies of B. burgdorferi sensu lato, even with those for which they are apparently not transmission competent (3, 5, 6, 9, 14, 15, 17). A possible explanation for this apparent paradox may be related to the fact that Borrelia genes are differentially expressed during the life cycle (4). While the gene expression of spirochetes residing in the tick’s midgut resembles that observed in culture, major outer surface proteins of Borrelia, including possible complement receptors, appear to be down-regulated in the salivary glands of ticks and within the vertebrate host (4). Thus, it is possible that complement taken up by the tick during the blood meal selectively kills spirochetes in the tick’s midgut (as seen in vitro), whereas spirochetes injected into the host by ticks whose salivary glands were infected prior to the uptake of blood may evade destruction. Studies to identify complement receptors on the spirochetes and to unveil the protective mechanisms of spirochetes against complement are under way.
Altogether, we conclude that complement plays a key role in the host-to-tick transmission of B. burgdorferi sensu lato and, as a consequence, in the global ecology of Lyme borreliosis.
Acknowledgments
We thank M. M. Simon, Freiburg, Germany, and A. P. Van Dam, Amsterdam, The Netherlands, for kindly providing spirochetal strains; S. Butcher, Oxford, United Kingdom, for providing horse sera; S. Fletcher, Auchtermuchty, United Kingdom, for providing deer sera; and D. Strange, Oxford, United Kingdom, for providing squirrel sera.
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