The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for the presence of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice treated with antibiotic were consistently culture negative, but tissues from some of the mice remained PCR positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, as determined based upon PCR results, and ticks from those cohorts transmitted spirochetes to naïve SCID mice, which became PCR positive but culture negative. Results indicated that following antibiotic treatment, mice remained infected with nondividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.

Transfer of immune serum from immunocompetent mice infected with B. burgdorferi protects mice against syringe challenge, and transfer of immune serum after infection is established induces arthritis resolution but does not clear infection or spirochetemia or resolve carditis. Immune serum had very-high-titer passive protective activity against syringe challenge but failed to protect mice against host-adapted spirochetes when they were challenged with infected tissue transplants. Mice were passively immunized at selected intervals relative to challenge inoculation with antisera to recombinant forms of an immunodominant region of flagellin, P39, and OspC (which are recognized by immune serum), but none provided protection or modified existing infection or disease. Results suggest that spirochetes within joints, but not in other tissues, are selectively vulnerable to immune serum and that immune serum appears to contain antibody against yet-to-be-identified antigens that may be selectively expressed in the context of joint tissue.

The population dynamics of Borrelia burgdorferi were quantified by real-time PCR targeting the flaB gene in skin (inoculation site, noninoculation site, and ear), heart (heart base and ventricle), quadriceps muscle, and the tibiotarsal joint at 1, 2, 4, 6, and 8 weeks after intradermal inoculation in C3H and C3H-scid mice. In addition, RNA transcription was assessed for several prototype genes, including flaB, ospA, ospC, dbpA, arp, vlsE, fbp, oppA-2, and p37-42. Spirochete numbers were equivalent in C3H and C3H-scid mice at 1 or 2 weeks and then declined in C3H mice, but they continued to rise and then plateaued in C3H-scid mice. Gene transcription was likewise higher in C3H-scid mice than in C3H mice, particularly at 4 or more weeks of infection. Gene transcription showed variation among tissues, with the highest levels of transcription in heart and joint tissue, which are sites of inflammation.Borrelia burgdorferi, the agent of Lyme disease, has a complex life cycle and must rapidly adapt to diverse microenvironments within resting ticks, feeding ticks, and mammalian or avian hosts. Aside from survival, these events must allow maintenance of the bacterium within the dormant tick, proliferation during tick feeding to enhance transmission, migration from the tick midgut to salivary glands during tick feeding, transmission into the host during tick feeding, dissemination within the host, immune evasion within the host, and, finally, successful acquisition by a new vector feeding upon an infected host. It is generally believed that these events are facilitated by the differential expression of selected genes. This has been best exemplified by a widely accepted paradigm of reciprocal expression and down-regulation of outer surface protein A (OspA) and OspC during tick feeding and transmission. OspA is abundantly expressed on spirochetes within unfed ticks (5), but is down-regulated during the course of feeding by the tick and entry into the mammalian host. In contrast, OspC appears to be expressed during feeding and transmission (19,50).Among other cues, these events have been shown to be triggered by changes in pH, cell density, temperature, and factors within the mammalian host (1, 13, 14, 20, 25, 37, 39, 40, 44-46, 49, 50, 52, 56). A recent study utilized whole-genome arrays to examine temperature-induced changes in B. burgdorferi and found that 215 open reading frames were differentially expressed at two different temperatures: 133 of them were expressed at temperatures greater than 35°C (40). Studies that have artificially mimicked the multiple conditions that take place in the feeding tick (elevated temperature, reduced pH, and increased cell density) have demonstrated the reciprocal down-regulation of OspA, P22, and Lp6.6 and up-regulation of OspC, decorin binding protein (DbpA), OspF, Mlp-8, and RpoS proteins (34, 55). OspE and some, but not all, of the OspE/F-related proteins (Erps) and OspE/F-like proteins (Elps) are also up-regulated under these conditions (29, 30, 51, 52). Furthermore, a number of B. ...

The agent of Lyme borreliosis, Borrelia burgdorferi, evades host immunity and establishes persistent infections in its varied mammalian hosts. This persistent biology may pose challenges to effective antibiotic treatment. Experimental studies in dogs, mice, and non-human primates have found persistence of B. burgdorferi DNA following treatment with a variety of antibiotics, but persisting spirochetes are non-cultivable. Persistence of B. burgdorferi DNA has been documented in humans following treatment, but the significance remains unknown. The present study utilized a ceftriaxone treatment regimen in the C3H mouse model that resulted in persistence of non-cultivable B. burgdorferi in order to determine their long-term fate, and to examine their effects on the host. Results confirmed previous studies, in which B. burgdorferi could not be cultured from tissues, but low copy numbers of B. burgdorferi flaB DNA were detectable in tissues at 2, 4 and 8 months after completion of treatment, and the rate of PCR-positive tissues appeared to progressively decline over time. However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues.

The effectiveness of a new first-in-class antibiotic, tigecycline (glycylcycline), was evaluated during the early dissemination (1 week), early immune (3 weeks), or late persistent (4 months) phases of Borrelia burgdorferi infection in C3H mice. Mice were treated with high or low doses of tigecycline, saline (negative-effect controls), or a previously published regimen of ceftriaxone (positive-effect controls). Infection status was assessed at 3 months after treatment by culture, quantitative ospA real-time PCR, and subcutaneous transplantation of joint and heart tissue into SCID mice. Tissues from all saline-treated mice were culture and ospA PCR positive, tissues from all antibiotic-treated mice were culture negative, and some of the tissues from most of the mice treated with antibiotics were ospA PCR positive, although the DNA marker load was markedly decreased compared to that in saline-treated mice. Antibiotic treatment during the early stage of infection appeared to be more effective than treatment that began during later stages of infection. The viability of noncultivable spirochetes in antibiotic-treated mice (demonstrable by PCR) was confirmed by transplantation of tissue allografts from treated mice into SCID mice, with dissemination of spirochetal DNA to multiple recipient tissues, and by xenodiagnosis, including acquisition by ticks, transmission by ticks to SCID mice, and survival through molting into nymphs and then into adults. Furthermore, PCR-positive heart base tissue from antibiotic-treated mice revealed RNA transcription of several B. burgdorferi genes. These results extended previous studies with ceftriaxone, indicating that antibiotic treatment is unable to clear persisting spirochetes, which remain viable and infectious, but are nondividing or slowly dividing.

Lymphadenopathy is a hallmark of acute infection with Borrelia burgdorferi, a tick-borne spirochete and causative agent of Lyme borreliosis, but the underlying causes and the functional consequences of this lymph node enlargement have not been revealed. The present study demonstrates that extracellular, live spirochetes accumulate in the cortical areas of lymph nodes following infection of mice with either host-adapted, or tick-borne B. burgdorferi and that they, but not inactivated spirochetes, drive the lymphadenopathy. The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT. Furthermore, nearly a quarter of hybridomas generated from regional lymph nodes during acute infection showed reactivity against a small number of recombinant Borrelia-antigens. Finally, neither the quality nor the magnitude of the B cell responses was altered in mice lacking the Toll-like receptor adaptor molecule MyD88. Together, these findings suggest a novel evasion strategy for B. burgdorferi: subversion of the quality of a strongly induced, potentially protective borrelia-specific antibody response via B. burdorferi's accumulation in lymph nodes.

C3H mice that were inoculated with ehrlichiae isolated from a patient with human granulocytic ehrlichiosis (HGE) developed anemia and leukopenia, but by day 24, they returned to normal values. Granulocytic morulae were present in peripheral blood and spleen smears on days 5 and 10, and there was a reduction in morulae on day 17. Ehrlichiae were present in HL-60 cell cultures of blood and spleen from all mice at all intervals. Pathogenicity, but not infectivity, waned with mouse passage but could be resurrected by SCID mouse passage. Various methods were tested for their relative sensitivity in detecting infection: blood smears, HL-60 cell cultures, polymerase chain reaction (PCR) amplification of a 16S recombinant DNA target, and a mouse infectivity assay. All assays detected the HGE agent in blood during early infection, but PCR and the mouse infectivity assay were most sensitive during late infection. Xenodiagnosis demonstrated that mice remain persistently infected through 55 days.

By using real-time quantitative PCR, the population dynamics and gene transcription of Borrelia burgdorferi were examined in ticks and skin of mice during acquisition of the infection from mice by ticks and during transmission of the infection from ticks to mice. Population dynamics were determined by using a flaB DNA target. A quantitative analysis of flaB, ospA, ospC, dbpA, and arp transcription was also performed. The results revealed that both uninfected larval and nymphal Ixodes scapularis ticks acquired B. burgdorferi as early as 1 day after attachment and that the sizes of spirochete populations within ticks increased during feeding. In addition, all gene targets revealed that there was RNA transcription during feeding. Similar events occurred within infected nymphal ticks feeding on uninfected hosts. Transmission from infected nymphal ticks to mice could be detected within 1 day after attachment. Analysis of skin during the first 3 days after attachment of infected ticks revealed rising numbers of spirochetes but minimal gene transcription. In contrast, the skin of mice with established infections revealed static populations of spirochetes and active but stable transcription of flaB, ospC, dbpA, and arp. There were consistent reductions in the number of spirochetes in the skin at the tick attachment sites compared to the number of spirochetes in the skin at nontick sites, but there were no differences in gene expression between tick and nontick skin sites. Evidence of ospA transcription in skin could be found 1 day after tick attachment but not thereafter.Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to and acquired from its hosts by Ixodes spp. hard ticks. The principal Ixodes vectors include Ixodes ricinus in Europe, Ixodes persulcatus in Eastern Europe and Asia, and Ixodes scapularis and Ixodes pacificus in North America (35,43). Acquisition and transmission are processes that involve the tick, the host, and the pathogen in reciprocal interactions with one other. After attachment, ticks induce host local inflammatory responses and immune responses against components within their saliva. To counteract these responses, tick saliva also contains substances that suppress or divert host immune responses (31,46).In addition to these vector and host processes, B. burgdorferi undergoes dynamic changes within the vector during both acquisition and transmission and within vertebrate hosts after transmission. These changes include striking variations in antigen expression. For example, B. burgdorferi is limited to the midgut of resting (unfed) nymphal and adult ticks (5) and usually express outer surface protein A (OspA) and very rarely OspC (18). After ticks attach and begin feeding, the spirochetes rapidly multiply (12), down-regulate or shed OspA, and up-regulate OspC during their migration to the salivary glands (41). Upon transmission, the spirochetes stay in the skin at the attachment site for several days and then disseminate throughout the vertebrate host (42). Early in infect...