Lyme borreliosis in rhesus macaques: effects of corticosteroids on spirochetal load and isotype switching of anti-borrelia burgdorferi antibody

Experimental Borrelia burgdorferi infection of rhesus monkeys is an excellent model of Lyme disease and closely parallels the infection in humans. Little is known about the interaction of host immunity with the spirochete in patients with chronic infection. We hypothesized that rapid development of anti-B. burgdorferi antibody in immunocompetent nonhuman primates (NHPs) is the major determinant of the reduction of the spirochetal load in Lyme borreliosis. This hypothesis was tested by measurement of the spirochetal load by PCR in association with characterization of the anti-B. burgdorferi humoral immune response in immunocompetent NHPs versus that in corticosteroid-treated NHPs. Although anti-B. burgdorferi immunoglobulin G (IgG) antibody was effectively inhibited in dexamethasone (Dex)-treated NHPs, anti-B. burgdorferi IgM antibody levels continued to rise after the first month and reached levels in excess of IgM levels in immunocompetent NHPs. This vigorous production of anti-B. burgdorferi IgM antibodies was also studied in vitro by measurement of antibody produced by B. burgdorferi-stimulated peripheral blood mononuclear cells. Despite these high IgM antispirochetal antibodies in Dex-treated NHPs, spirochetal loads were much higher in these animals. These data indicate that Dex treatment results in interference with isotype switching in this model and provide evidence that anti-B. burgdorferi IgG antibody is much more effective than IgM antibody in decreasing the spirochetal load in infected animals.     

Protective niche for Borrelia burgdorferi to evade humoral immunity

The Lyme disease spirochete, Borrelia burgdorferi, is an extracellular microbe that causes persistent infection despite the development of strong immune responses against the bacterium. B. burgdorferi expresses several ligand-binding lipoproteins, including the decorin-binding proteins (Dbps) A and B, which may mediate attachment to decorin, a major component of the host extracellular matrix during murine infection. We show that B. burgdorferi was better protected in the joints and skin, two tissues with a higher decorin expression, than in the urinary bladder and heart, two tissues with a lower decorin expression, during chronic infection of wild-type mice. Targeted disruption of decorin alone completely abolished the protective niche in chronically infected decorin-deficient mice but did not affect the spirochete burden during early infection. The nature of protection appeared to be specific because the spirochetes with higher outer surface protein C expression were not protected while the protective niche seemed to favor the spirochetes with a higher dbpA expression during chronic infection. These data suggest that spirochetal DbpA may interact with host decorin during infection and such interactions could be a mechanism that B. burgdorferi uses to evade humoral immunity and establish chronic infection.     

Heritable susceptibility to severe Borrelia burgdorferi-induced arthritis is dominant and is associated with persistence of large numbers of spirochetes in tissues.

In human Lyme disease, symptoms with widely varying levels of severity have been observed. A mouse model of Lyme disease has been developed which allows analysis of mice with mild, moderate, and severe pathologies after inoculation with the spirochete Borrelia burgdorferi. To determine whether the differences in symptoms reflect differences in the number of spirochetes persisting in affected tissues, a sensitive PCR technique was developed to detect B. burgdorferi DNA in virtually any tissue of an infected mouse. This analysis, which detects DNA from as few as three spirochetes, revealed the presence of B. burgdorferi DNA in many tissues from severely arthritic C3H/HeJ mice as early as 1 week postinfection. The heart, ear, and ankle were particularly heavily infected, although B. burgdorferi DNA was also detected in spleen, liver, brain, kidney, bladder, uterus, and lymph nodes. In contrast, much lower levels of spirochete DNA were detected in tissues of infected BALB/c mice, which develop less severe arthritis when infected with B. burgdorferi than do C3H/HeJ mice. This difference was evident throughout the 5-week analysis. A competitive PCR method allowed determination of the absolute number of spirochete gene sequences in infected tissues. Ankles and hearts from C3H/HeJ mice were found to harbor 10(7) copies of the B. burgdorferi ospA gene, while these tissues from BALB/c mice contained 5- and 10-fold less B. burgdorferi DNA, respectively. The genetic regulation of severe pathology was analyzed by infecting the offspring of a cross between C3H/HeJ and BALB/c mice. The F1 mice developed severe arthritis and contained high levels of Borrelia DNA in the heart and ankle, similar to the C3H/HeJ parent. These findings indicate that susceptibility to severe arthritis is a dominant trait and suggest that it may correlate with high levels of persisting spirochetes. Models of pathology in Lyme disease should take into consideration the fact that severity of pathology may be directly related to the number of organisms in infected tissues.     

Increasing the interaction of Borrelia burgdorferi with decorin significantly reduces the 50 percent infectious dose and severely impairs dissemination

Tight regulation of surface antigenic expression is crucial for the pathogenic strategy of the Lyme disease spirochete, Borrelia burgdorferi. Here, we report the influence of increasing expression of decorin-binding protein A (DbpA), one of the most investigated spirochetal surface adhesins, on the 50% infectious dose (ID(50)), dissemination, tissue colonization, pathogenicity, and persistence of B. burgdorferi in the murine host. Our in vitro assays showed that increasing DbpA expression dramatically increased the interaction of B. burgdorferi with decorin and sensitivity to growth inhibition/killing by anti-DbpA antibodies; however, this increased interaction did not affect spirochetal growth and replication in the presence of decorin. Increasing DbpA expression significantly reduced ID(50) values and severely impaired dissemination in severe combined immunodeficiency (SCID) and immunocompetent mice. During infection of SCID mice, B. burgdorferi with increased DbpA expression was able to effectively colonize heart and skin tissues, but not joint tissues, completely abrogating arthritis virulence. Although increasing DbpA expression did not affect spirochetal persistence in the skin, it diminished the ability of B. burgdorferi to persist in the heart and joint tissues during chronic infection of immunocompetent mice. Taken together, the study highlights the importance of controlling surface antigen expression in the infectivity, dissemination, tissue colonization, pathogenicity, and persistence of B. burgdorferi during mammalian infection.     

Cystitis induced by infection with the Lyme disease spirochete, Borrelia burgdorferi, in mice.

Previous studies have demonstrated that the urinary bladder is a consistent source for isolating the Lyme disease spirochete, Borrelia burgdorferi, from both experimentally infected and naturally exposed rodents. We examined histopathologic changes in the urinary bladder of different types of rodents experimentally infected with Lyme spirochetes, including BALB/c mice (Mus musculus), nude mice (M. musculus), white-footed mice (Peromyscus leucopus), and grasshopper mice (Onychomys leucogaster). Animals were inoculated intraperitoneally, subcutaneously, or intranasally with low-passaged spirochetes, high-passaged spirochetes, or phosphate-buffered saline. At various times after inoculation, animals were killed and approximately one-half of each urinary bladder and kidney were cultured separately in BSK-II medium while the other half of each organ was prepared for histologic examination. Spirochetes were cultured from the urinary bladder of all 35 mice inoculated with low-passaged spirochetes while we were unable to isolate spirochetes from any kidneys of the same mice. The pathologic changes observed most frequently in the urinary bladder of the infected mice were the presence of lymphoid aggregates, vascular changes, including an increase in the number of vessels and thickening of the vessel walls, and perivascular infiltrates. Our results demonstrate that nearly all individuals (93%) of the four types of mice examined had a cystitis associated with spirochetal infection.     

Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans.

Current biological and serological techniques for demonstrating infections by Borrelia burgdorferi can be inconclusive. In order to monitor Lyme borreliosis, we developed a rapid and sensitive assay for B. burgdorferi antigens in infected hosts. Polyclonal rabbit antisera were raised against membrane vesicles and an 83-kDa vesicle-associated protein band that was purified from in vitro B. burgdorferi cultures. Immunoglobulin G (IgG) antibodies were recovered from these sera and tested for a species-specific reaction with several geographically diverse Borrelia isolates by immunoblot analysis. Parlodion-coated electron microscope grids were activated with anti-vesicle F(ab')2 fragments and then incubated with confirmed or experimental sources of spirochetal antigens. Such sources included cultured spirochetes; spirochete culture supernatants; samples of urine, blood, or serum from mice, dogs, and humans; triturates of Ixodes ticks; and bladder, spleen, liver, kidney, heart, or brain tissues from infected or control mice. Captured antigens were assayed by immune electron microscopy by using anti-83-kDa IgG antibodies and protein A-colloidal gold conjugates. The results indicated that B. burgdorferi appears to shed surface antigens which are readily detectable in urine, blood, and several organs from infected hosts. Such antigens were detectable in mouse urine at dilutions exceeding 10(-6). Intact spirochetes were frequently observed on grids incubated with blood, spleen, or bladder preparations, and B. burgdorferi was reisolated from the urinary bladders of all experimentally infected mice. These results indicated that B. burgdorferi antigens arise in a variety of host materials. Such antigens can be captured and identified with specific polyclonal antibodies, providing a sensitive assay for monitoring and studying Lyme borreliosis.     

Persistent cardiac and urinary tract infections with Borrelia burgdorferi in experimentally infected Syrian hamsters.

The heart can be severely affected in humans with Lyme disease, causing conduction defects and, rarely, heart failure. Although immunodeficient and young mice may develop cardiac lesions, cultivation of Borrelia burgdorferi from cardiac tissues of experimentally infected animals has not been reported previously. We infected Syrian hamsters with B. burgdorferi 297 and found a marked tropism of the spirochete for myocardial and urinary tract tissues. Fifty-six of 57 hearts (98%) and 52 of 58 bladders (90%) were culture positive. The cardiac infection was persistent and could be documented in 21 of 22 hearts (96%) cultured from days 28 to 84 postinfection. The urinary tract was also a site of persistent infection in most animals, with 18 of 23 bladders (78%) being culture positive from days 28 to 84. The persistence of spirochetes was specific for the heart and bladder, as indicated by negative cultures of specimens from the liver and spleen, in which only 1 of 23 cultures was positive from days 28 to 84. Because of the high isolation rates, tropism, and persistence that we found for B. burgdorferi in the hamster heart and bladder, these sites will be useful and important for the cultivation of spirochetes in experimental studies that evaluate the efficacies both of candidate vaccines in preventing infection and of antibiotics in eradicating organisms from privileged sites. In addition, the clear demonstration of persistent cardiac infection with B. burgdorferi may provide a useful model for studying the pathogenesis of cardiac Lyme disease.     

Cultivation at 37°C enhances Borrelia burgdorferi sensu stricto infectivity for hamsters

Borrelia burgdorferi sensu stricto (s.s.), the causative agent of Lyme disease in North America is transmitted to the mammalian host by ticks belonging to the genus, Ixodes. Antibodies to several spirochetal proteins, most notably outer surface protein C (OspC), have been observed in early infection in both humans and laboratory animals. Thus, the expression of these proteins have been postulated to play a role in tick transmission and spirochetal infectivity for the mammalian host. B. burgdorferi strain JMNT was induced to produce increased levels of OspC by cultivation in BSK medium at 37 degrees C. To diminish expression of OspC, spirochetes were cultivated at 31 degrees C. Spirochetes shifted down from 37 degrees C to 31 degrees C or up from 31 degrees C to 37 degrees C for 1 week contained equivalent amounts of OspC. To evaluate spirochetal infectivity, hamsters were inoculated subcutaneously with 1 x 10(4) or 1 x 10(6) spirochetes grown at the above-mentioned temperatures. Hamsters inoculated with spirochetes expressing high amounts of OspC all became infected, irrespective of the inoculum size. None of the hamsters inoculated with 1 x 10(4) spirochetes grown at 31 degrees C or in cultures shifted down from 37 degrees C to 31 degrees C were infected. All infected hamsters, confirmed by isolation of spirochetes in ear and/or bladder cultures, had an antibody response to OspC. In contrast, all non-infected hamsters lacked antibodies to OspC. We conclude that cultivation of spirochetes at 37 degrees C enhances their infectivity for hamsters. This study also suggests there is a correlation between enhancement of OspC expression and spirochetal infectivity for hamsters.     

Borrelia burgdorferi in Tick Cell Culture Modulates Expression of Outer Surface Proteins A and C in Response to Temperature

The Lyme disease spirochete Borrelia burgdorferi sensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding. The switching of these proteins correlates with increased spirochetal infectivity for the mammal. We examined the effect of temperature on differential expression of OspA and OspC by B. burgdorferi cocultivated with a cell line isolated from the vector tick Ixodes scapularis. The effect of incubation at 31, 34, or 37 degrees C on expression of OspA and OspC by B. burgdorferi JMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting. The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31 degrees C and declined with increasing temperature in both strains. OspC production was enhanced in spirochetes cocultivated with tick cells at 37 degrees C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37 degrees C, but OspA content was not appreciably affected by temperature. Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.     

Antibody to a 39-kilodalton Borrelia burgdorferi antigen (P39) as a marker for infection in experimentally and naturally inoculated animals.

Borrelia burgdorferi expresses a conserved, species-specific 39-kDa protein (P39) that can stimulate antibodies during human infection. To confirm that anti-P39 antibodies are produced consistently in animals exposed to infectious spirochetes, white-footed mice, Peromyscus leucopus, and laboratory white mice, Mus musculus (strain BALB/c), were experimentally inoculated with either infectious or noninfectious B. burgdorferi and the antibody response to P39 was determined by immunoblot at 21 days postinoculation. All mice inoculated with approximately 10(7) infectious B. burgdorferi produced anti-P39 antibodies and were cultured positive for this spirochete. Mice inoculated with similar numbers of inactivated or viable noninfectious B. burgdorferi still producing P39 did not induce anti-P39 antibodies. By contrast, putative antiflagellin antibodies were detected in less than 18% of the infected animals, which supports the notion that antibody reactive with flagellin may not be reliable as a marker for B. burgdorferi exposure as was originally thought. Mice infected with B. burgdorferi following exposure to ticks (Ixodes dammini) produced anti-P39 antibodies no later than 7 days postinfection, indicating that P39 is an effective immunogen in natural infections. Notably, anti-P39 antibodies were the predominant B. burgdorferi reactive antibodies detected early in the infection. Our results indicate that anti-P39 antibodies are produced in response to an active infection and are therefore reliable markers for infection in experimentally and naturally inoculated animals.     

Culturing selects for specific genotypes of Borrelia burgdorferi in an enzootic cycle in Colorado.

In Colorado, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease, is maintained in an enzootic cycle between Ixodes spinipalpis ticks and Neotoma mexicana rats (27). The frequencies of flagellin (fla), 66-kDa protein (p66), and outer surface protein A (ospA) alleles were examined in 71 B. burgdorferi isolates from samples from Colorado. Approximately two-thirds of these samples were isolates from I. spinipalpis ticks that had been cultured in BSK-H medium prior to DNA extraction. The remaining samples were from total DNA extracted directly from infected I. spinipalpis ticks. A portion of each gene was amplified by PCR and screened for genetic variability by single-strand conformation polymorphism (SSCP) analysis. We identified three alleles in the fla gene, seven in the p66 gene, and seven in the ospA gene. Sequencing verified that the amplified products originated from B. burgdorferi template DNA and indicated 100% sensitivity and specificity of the SSCP analysis. The frequencies of the p66 and ospA alleles were significantly different between cultured and uncultured spirochetes. The number of three-locus genotypes and the genetic diversity of alleles at all loci were consistently lower in cultured spirochetes, suggesting that culturing of B. burgdorferi in BSK-H medium may select for specific genotypes.     

A guinea pig model for Lyme disease.

We report that outbred Hartley guinea pigs are susceptible to Borrelia burgdorferi. We recovered spirochetes from 57 of 60 (95%) guinea pigs inoculated when < or = 3 months of age. In contrast, animals inoculated when > or = 6 months of age were resistant to infection as defined by recovery of organisms at > or = 4 weeks postinoculation. Infection was widely disseminated: B. burgdorferi was recovered from 83% of bladders, 64% of knee joints, 57% of hearts, 48% of spleens, and 38% of spinal cords examined within 4 weeks of inoculation. Histopathologic changes were common in the heart (88%) (preferential involvement of perineural tissues near the annulus fibrosus) and bladder (76%) and were also noted in a minority of spinal cords (13%) and knee joints (9%). Western immunoblots demonstrated an immunoglobulin G response to B. burgdorferi, particularly to the 24-, 31- (OspA), 39-, and 41-kDa (flagellin) antigens. Infection was cleared from most tissues with the passage of time; spirochetes were recovered from 63% of tissues removed from guinea pigs at < or = 4 weeks after inoculation but from only 32% at > or = 8 weeks postinoculation (P < 0.001). An exception was the failure to clear spirochetes from infected knees, 90% of which were culture positive even when evaluated at > or = 8 weeks postinoculation. The guinea pig provides a new model useful for studying host-spirochete interactions in Lyme disease.     

Comparison of polymerase chain reaction and culture for detection of Borrelia burgdorferi in naturally infected Peromyscus leucopus and experimentally infected C.B-17 scid/scid mice.

Culture and the polymerase chain reaction (PCR) were compared for detection of Borrelia burgdorferi infection in wild-caught Peromyscus leucopus and experimentally inoculated C.B-17 scid/scid (severe combined immunodeficient) mice. PCR targeted highly conserved regions of the ospA gene and could detect one to five cultured organisms and 10 to 50 copies of molecularly cloned ospA DNA. Organs (kidney, spleen, and urinary bladder) and/or ear biopsy samples were obtained from 108 captured P. leucopus mice, and tissues were obtained from 7 experimentally inoculated mice. A simple sample-processing procedure with proteinase K and detergent treatment was used in the PCR analysis. Overall, B. burgdorferi was detected in 29 of 108 (27%) P. leucopus mice by culture and in 31 of 108 (29%) mice by PCR. As assessed by the kappa statistic, agreement between PCR and culture was high for ear and bladder (kappa = 0.80 and 0.65, respectively) and low for kidney and spleen (kappa = 0.37 and 0.03, respectively). While concordant results were obtained from 98 animals, PCR detected B. burgdorferi from 6 additional mice for which cultures were negative and culture detected B. burgdorferi from 4 animals which were PCR negative. Further phenol-chloroform extraction of DNA in a limited number of samples improved the sensitivity of PCR compared with that of culture. These results indicate that PCR may be as sensitive as culture for detecting B. burgdorferi in ear samples and that PCR analysis is suitable for establishing the infection status of animals in mark-release-recapture studies.     

Antibody response in white-footed mice (Peromyscus leucopus) experimentally infected with the Lyme disease spirochete (Borrelia burgdorferi).

White-footed mice (Peromyscus leucopus), the primary reservoir for Borrelia burgdorferi in the northern midwest and northeastern United States, were experimentally inoculated with an infectious strain or a noninfectious strain of the Lyme disease spirochete and examined for their specific antibody response with the enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Immunoglobulin M (IgM) anti-B. burgdorferi antibodies were detected in mice 1 to 2 days after inoculation with either the infectious or noninfectious strain of spirochetes and peaked on days 4 and 5. Mice inoculated with the infectious strain of spirochete had a secondary increase in IgM 21 days after inoculation. Mice also produced both IgG1 and IgG2 antibodies beginning 5 to 7 days after inoculation and they increased in titer until 84 days after inoculation when the experiment was terminated. Western blot analysis of sequential plasma samples from mice inoculated with the infectious strain of spirochete demonstrated the development of IgM, IgG1, and IgG2 antibodies to numerous spirochetal antigens, whereas mice inoculated with the noninfectious strain had reduced blot patterns with antibodies reactive primarily to the 31,000-kilodalton outer surface protein A. Persistent spirochetal infection in some mice, in spite of a strong and diverse antibody response, deserves further investigation.     

Cardiac involvement in non-human primates infected with the Lyme disease spirochete Borrelia burgdorferi

To investigate cardiac involvement in the non-human primate (NHP) model of Lyme disease, we inoculated 39 adult Macaca mulatta with Borrelia burgdorferi sensu stricto strains N40 (BbN40) by needle (N=22, 14 immunocompetent (IC), seven permanently immunosuppressed (IS), and four transiently immunosuppressed (TISP)) or by tick-bite (N=4, all TISP) or strain 297 (Bb297) by needle (N=2 IS), or with B. garinii strains Pbi (N=4, 2 TISP and 2 IS), 793 (N=2, TISP) or Pli (N=2, TISP). Five uninfected NHPs were used as controls. Infection and inflammation was studied in the hearts and the aorta removed at necropsy 2-32 months after inoculation by (1) H&E and trichrome-staining; (2) immunohistochemistry and digital image analysis; (3) Western blot densitometry; and (4) TaqMan RT-PCR. All NHPs inoculated with BbN40 became infected and showed carditis at necropsy. The predominant cells were T cells, plasma cells, and macrophages. There was increased IgG and IgM in the heart independent of immunosuppression. The B-cell chemokine BLC was significantly increased in IS-NHPs. There was increased deposition of the complement membrane attack complex (MAC) in TISP and IS-NHPs. The spirochetal load was very high in all BbN40-inoculated IS-NHPs but minimal if any in IC or TISP NHPs. Double-immunostaining revealed that many spirochetes in the heart of BbN40-IS NHPs had MAC on their membranes. We conclude that carditis in NHPs infected with B. burgdorferi is frequent and can persist for years but is mild unless they are immunosupressed.     

Lyme Borreliosis in Rhesus Macaques: Effects of Corticosteroids on Spirochetal Load and Isotype Switching of Anti-Borrelia burgdorferi Antibody

Experimental Borrelia burgdorferi infection of rhesus monkeys is an excellent model of Lyme disease and closely parallels the infection in humans. Little is known about the interaction of host immunity with the spirochete in patients with chronic infection. We hypothesized that rapid development of anti-B. burgdorferi antibody in immunocompetent nonhuman primates (NHPs) is the major determinant of the reduction of the spirochetal load in Lyme borreliosis. This hypothesis was tested by measurement of the spirochetal load by PCR in association with characterization of the anti-B. burgdorferi humoral immune response in immunocompetent NHPs versus that in corticosteroid-treated NHPs. Although anti-B. burgdorferi immunoglobulin G (IgG) antibody was effectively inhibited in dexamethasone (Dex)-treated NHPs, anti-B. burgdorferi IgM antibody levels continued to rise after the first month and reached levels in excess of IgM levels in immunocompetent NHPs. This vigorous production of anti-B. burgdorferi IgM antibodies was also studied in vitro by measurement of antibody produced by B. burgdorferi-stimulated peripheral blood mononuclear cells. Despite these high IgM antispirochetal antibodies in Dex-treated NHPs, spirochetal loads were much higher in these animals. These data indicate that Dex treatment results in interference with isotype switching in this model and provide evidence that anti-B. burgdorferi IgG antibody is much more effective than IgM antibody in decreasing the spirochetal load in infected animals.