Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi

We were unable to demonstrate the presence of the classic enterobacterium-type lipopolysaccharide in the cells of the Lyme disease spirochete, Borrelia burgdorferi B31. This finding was primarily based on chemical analysis and the absence of free lipid A upon mild acid hydrolysis of the appropriate cell extracts. These results do not preclude the possible existence of an unusual lipopolysaccharide-like compound(s) in B. burgdorferi.     

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.     

Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete.

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in hypertonic sucrose followed by isopycnic ultracentrifugation of the plasmolyzed cells. The unilamellar vesicles thus obtained were shown to be OMs by the following criteria: (i) they contained OspA and OspB; (ii) they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture electron microscopy was identical to that of OMs of intact organisms. Consistent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small proportions of the total cellular content of OspA or OspB were OM associated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [3H]palmitate or 35S-amino acids demonstrated that the OMs contained both nonlipidated and lipidated proteins. This fractionation procedure was also used to isolate OMs from virulent and avirulent isolates of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluorography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of antibody reactivity was directed against intracellular proteins. The availability of isolated OMs should facilitate efforts to elucidate the complex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.     

Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi.

Previous studies have demonstrated that Borrelia burgdorferi in the midguts of infected ticks shows increased expression of the antigenic outer surface protein OspC after the ticks have ingested a blood meal. This differential expression is at least partly due to a change in temperature, as an increase in OspC levels is also observed when cultures are shifted from 23 to 35 degrees C. Immunoblotting of bacterial lysates with sera from infected mice indicated that the levels of several additional antigens were also increased in bacterial cultures shifted to 35 degrees C; we have identified one antigen as OspE. We have also observed differential expression of OspF, which has been proposed to be coexpressed in an operon with the gene encoding OspE.     

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 disease spirochete, Borrelia burgdorferi, endemic in epicenter at Turkey Point, Ontario

The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt, and Brenner, was discovered in blacklegged ticks, Ixodes scapularis Say at Turkey Point, Ontario, Canada. We report the first isolation of B. burgdorferi from a vertebrate animal collected on mainland Ontario. During this 2-yr study, spirochetes were isolated from the white-footed mouse, Peromyscus leucopus Rafinesque, and attached I. scapularis larvae. Similarly, isolates of B. burgdorferi were cultured from blacklegged tick adults, and confirmed positive with polymerase chain reaction by targeting OspA and rrf (5S) -rrl (23S) genes. Moreover, all isolates of B. burgdorferi from this area had complementary genetic structure, and the second primer set amplicons confirmed the first primer set amplification products. These findings show an epicenter endemic for B. burgdorferi within an established population of I. scapularis at Turkey Point.     

Lyme disease spirochete,Borrelia burgdorferi endemic at epicenter in Rondeau Provincial Park,Ontario

The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt, and Brenner was discovered in blacklegged ticks, Ixodes scapularis Say at Rondeau Provincial Park, Ontario, Canada During this 2-yr study, spirochetes were found in B. burgdorferi-positive I. scapularis larvae attached to B. burgdorferi-infected white-footed mice, Peromyscus leucopus Rafinesque. Isolates of B. burgdorferi were cultured from blacklegged tick adults, and confirmed positive with polymerase chain reaction by targeting OspA and rrf (5S)-rrl (23S) genes. These findings show an endemic area for B. burgdorferi within an established population of L. scapularis at Rondeau Provincial Park.     

Serologic surveillance for the Lyme disease spirochete, Borrelia burgdorferi, in Minnesota by using white-tailed deer as sentinel animals.

To determine the effectiveness of white-tailed deer as sentinel animals in serologic surveillance programs for Borrelia burgdorferi, we performed enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting analyses on 467 deer serum samples. The seropositivity rate in the ELISA was 5% for the 150 samples collected at the three sites in which the tick Ixodes scapularis was absent. The three sites with established I. scapularis populations had a seropositivity rate of 80% for 317 samples. Results were similar for two closely situated sites, one with an established I. scapularis population and one without; these sites were only 15 km apart. Rates of seropositivity were significantly higher in yearling and adult deer than in fawns. The mean numbers of bands seen on Western immunoblots were 3.0 for samples negative in the ELISA and 13.8 for samples positive in the ELISA; all of these samples were collected from sites in which I. scapularis was established. At sites in which I. scapularis was absent, the mean numbers of bands seen were 1.6 for samples negative in the ELISA and 8.2 for samples positive in the ELISA. There were 14 different B. burgdorferi antigens that reacted with more than 50% of the ELISA-positive samples from areas with I. scapularis. A 19.5-kDa antigen reacted with 94% of the ELISA-positive samples. Reactivity against OspA and OspB was weak a infrequent (2%). Serologic analysis of white-tailed deer sera appears to be an accurate and sensitive surveillance method for determining whether B. burgdorferi is present in specific geographic locations.     

Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent.

A simple procedure for extraction of plasmid-enriched DNA from borreliae was used in a plasmid analysis of 13 strains of the Lyme disease agent, Borrelia burgdorferi. The extracted DNA was subjected to low-percentage agarose gel electrophoresis and examined either directly by ethidium bromide staining or after hybridization of the plasmids in situ with a DNA probe for the gene encoding the major outer membrane protein OspA. Each isolate had four to seven discernible plasmids of various sizes. Only 2 of the 13 strains had the same plasmid profile. The ospA gene probe hybridized to large plasmids to strains from both North America and Europe. A strain which had been passaged many times was found to have lost two of the six plasmids originally present. These findings indicate the potential usefulness of plasmid analysis as a strain-typing procedure and for identifying possible plasmid-conferred virulence factors.     

LuxS-mediated quorum sensing in Borrelia burgdorferi,the lyme disease spirochete

The establishment of Borrelia burgdorferi infection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues. This complex process requires regulated synthesis of many bacterial proteins. We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete. The luxS gene of B. burgdorferi was identified and demonstrated to encode a functional enzyme by complementation of an Escherichia coli luxS mutant. Cultured B. burgdorferi responded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins. Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes.     

Differential infectivity of the Lyme disease spirochete Borrelia burgdorferi derived from Ixodes scapularis salivary glands and midgut

Blood fed nymphal Ixodes scapularis Say infected with Borrelia burgdorferi were dissected to obtain salivary gland and midgut extracts. Extracts were inoculated into C3H/HeJ mice, and ear, heart, and bladder were cultured to determine comparative infectivity. Aliquots of extracts were then analyzed by quantitative polymerase chain reaction to determine the number of spirochetes inoculated into mice. A comparative median infectious dose (ID50) was determined for both salivary gland and midgut extract inoculations. Our data demonstrated a statistically significant difference (P < 0.002) in the ID50 derived from salivary gland (average = 18) versus midgut (average = 251) extracts needed to infect susceptible mice. A rationale for the differential infectivity of salivary and midgut derived spirochetes is discussed.     

Lyme disease assay which detects killed Borrelia burgdorferi.

We developed an in vitro assay showing that Borrelia burgdorferi organisms were killed by serum from patients with Lyme disease. Twenty of 20 Lyme disease serum samples caused B. burgdorferi killing in a range of 36 to 99% compared with the mean number of viable spirochetes when sera from 10 healthy individuals were used. The percentage of killing of B. burgdorferi increased with convalescent serum from patients with early Lyme disease. The borreliacidal activity was detectable in some sera diluted 640-fold and was abrogated after treatment with anti-human immunoglobulin G. In contrast, pooled or individual normal human serum did not cause a decrease in the number of viable B. burgdorferi. Borreliacidal activity was also not detected in sera from patients with relapsing fever, rocky mountain spotted fever, syphilis, mononucleosis, rheumatoid factor, or DNA antibodies. Our results show that borreliacidal activity can be used as a specific serodiagnostic test for detecting Lyme disease.     

Lyme disease and Borrelia burgdorferi infections in Europe

Lyme disease is endemic in Europe. The strains of the causative agent, Borrelia burgdorferi, seem to be antigenically more heterogeneous than the North American isolates. The only documented vector for this bacterium in Europe is ixodes ricinus, but other vectors might be involved as observed in the United States. The tick hosts are not yet well documented in Europe. Human infection occurs principally during summer months. The clinical aspect of the disease has particular features in Europe: at the early stage of the disease, a single and large erythema chronicum migrans is observed on the skin; complications often include meningoradiculonevritis (Bannwarth's syndrome) and later, acrodermatitis chronica atrophians; arthritis is less frequent in Europe than in the USA. The culture of B. burgdorferi from the lesions is difficult. The diagnosis of the disease is performed on the basis of serological tests: immunofluorescence assay where the important thing is to define a cutoff titer; ELISA tests using either whole cells or supernatant of sonicated cells or flagellar antigen; passive haemagglutination for IgG; IgM solid phase haemadsorption; Western blot (immunoblot) seems interesting to perform on a research basis to determine to which protein antigens patients are responding with antibody. Once antibody production begins, it is usually in the form of IgM antibody to flagellin protein (41 kD), with time, both IgM and IgG antibodies to a variety of other antigens appear. Prophylaxis is based on health services and public education because a prompt removal of the tick diminishes risk of infection with B. burgdorferi (4 p. cent of cases after tick bite). The treatment includes aminopenicillins or tetracyclines at the early stage. The second and third stages of borreliosis are treated by high doses aminopenicillins or cetriaxone.     

Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation.

In vitro cultivation of Borrelia burgdorferi, the etiologic agent of Lyme spirochetosis, allows for the isolation and growth of this bacterium from infected tissues. However, continuous cultivation in modified Kelly medium causes a reduction in the number of detectable plasmids and the loss of infectivity in the white-footed mouse, Peromyscus leucopus. In an unpassaged culture of B. burgdorferi, nine plasmids were present, including seven linear plasmids ranging in size from 49 to 16 kilobases (kb) and two circular plasmids of 27 and 7.6 kb. The 7.6-kb circular and 22-kb linear plasmids were no longer detectable in spirochetes noninfective in white-footed mice, suggesting that a gene(s) encoding for factors responsible for infection may be present on one or more of these extrachromosomal elements. Furthermore, changes in spirochetal proteins and lipopolysaccharide-like material were observed also during early cultivation and may be related to loss of infectivity.     

Demonstration of a B-lymphocyte mitogen produced by the Lyme disease pathogen, Borrelia burgdorferi.

Lyme disease refers to the multisymptomatic illness in humans which results from infection with the tick-borne spirochete Borrelia burgdorferi. The white-footed mouse is the major reservoir for B. burgdorferi and, upon infection, certain inbred mice develop symptoms similar to those reported in human disease. Sonicated preparations of washed spirochetes were found to have potent mitogenic activity when cultured with lymphocytes from naive C57BL/6, C3H/HeJ, or BALB/c mice. The activity of the B. burgdorferi sonicate was approximately fourfold greater than that of a similarly prepared Escherichia coli sonicate. Polymyxin B efficiently inhibited the mitogenic activity of the E. coli sonicate but only slightly inhibited that of the B. burgdorferi sonicate, suggesting that a lipid A-containing lipopolysaccharide was not responsible for the B. burgdorferi activity. Kinetic analysis indicated peak proliferation at 2 to 3 days of culturing, suggesting polyclonal activation. B- and T-lymphocyte depletion experiments indicated that the major cell type responding to the B. burgdorferi mitogen was the B lymphocyte. This mitogen stimulated murine B cells not only to proliferate but also to differentiate into antibody-secreting cells, as demonstrated by the production of immunoglobulin by stimulated splenocytes. Furthermore, the sonicated preparation stimulated the B-cell tumor line CH12.LX to secrete immunoglobulin in the absence of accessory cells. B. burgdorferi also stimulated interleukin-6 production in splenocyte cultures. The observation that B. burgdorferi can stimulate activation of and immunoglobulin production by normal B lymphocytes may directly reflect on the development of arthritis associated with persistent infection by this organism.     

Experimental acquisition of the Lyme disease spirochete, Borrelia burgdorferi, by larval Ixodes dammini (Acari: Ixodidae) during partial blood meals

The duration of feeding by larval Ixodes dammini was directly correlated with the proportion of larvae acquiring infection with the Lyme disease spirochete. The infection rate of ticks attached to hosts for 8 h (1.4%) was much lower than ticks feeding to repletion (87.5%). Infection was also detected in partially fed larvae dropping off dead animals. These partially fed larvae successfully reattached to rodents, fed to repletion, and molted to nymphs. Partially fed larvae did not transmit Borrelia burgdorferi when refeeding as larvae, but the resultant nymphs were capable of transmitting spirochetes. Infected host-seeking larvae collected in nature cannot be judged a priori to have acquired B. burgdorferi transovarially.     

Infectivity of the highly transformable BBE02- lp56- mutant of Borrelia burgdorferi, the Lyme disease spirochete, via ticks

Infectious Borrelia burgdorferi strains that have increased transformability with the shuttle vector pBSV2 were recently constructed by inactivating the gene encoding BBE02, a putative restriction-modification gene product expressed by the linear plasmid lp25 (Kawabata et al., Infect. Immun. 72:7147-7154, 2004). The absence of the linear plasmid lp56, which carries another putative restriction-modification gene, further enhanced transformation rates. The infectivity of these mutants was assessed previously in mice that were inoculated with needle and syringe and was found to be equivalent to that of wild-type spirochetes. Here we examined the infectivity of spirochetes to ticks after capillary inoculation of Ixodes scapularis nymphs and the subsequent spirochetal infectivity to mice via ticks by using B. burgdorferi B31 clonal isolates lacking lp56 and/or BBE02. The absence of lp56 (but not BBE02) correlated with a lower number of spirochetes in ticks after feeding on mice; this plasmid thus may play a role, albeit not an essential one, in supporting spirochetal survival in the feeding tick. Importantly, however, the absence of lp56 and BBE02 did not detectably influence infectivity to mice via ticks.     

Serodiagnosis of Lyme disease by ELISA using Borrelia burgdorferi flagellum antigen

Antibodies to a 41,000 (41 kD) polypeptide in flagella of Borrelia burgdorferi were measured in patients with Lyme disease in Japan by flagellum ELISA. The IgG and IgM Classes of antibodies to a flagellum antigens were detected in the sera as early as 0.5 months after infection. The IgG antibodies continued to exist in their sera for more than one year, while the IgM antibodies quickly faded out from their sera. With respect to a diagnostic specificity of the flagellum ELISA, false positive reactions showing more than 10% were observed in sera with high levels of IgG or IgM, and with anti-syphilis antibody. This method, however, was unaffected by sera with high levels of IgA, rheumatoid factor or anti-nuclear antibody. In three cases of patients with erythema migrans preceded by tick-bite, and treated with antibiotics, seronegative results were observed by a immunoperoxidase (IP) test. Since two of them showed the positive level of IgM antibody by the flagellum ELISA, this method seems to be more sensitive and useful than the IP test for serodiagnosis of the Lyme disease.