Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii

It has been known for decades that human Lyme disease is caused by the three spirochete species Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Recently, Borrelia valaisiana, Borrelia spielmanii, and Borrelia bissettii have been associated with Lyme disease. We report the complete genome sequences of B. valaisiana VS116, B. spielmanii A14S, and B. bissettii DN127.     

Acylated proteins in Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae.

Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae produced several lipoproteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of bacteria grown in [3H]palmitate. Five major acylated proteins were demonstrated by sequential alkaline and acid hydrolysis. High-pressure liquid chromatography of isolated proteins confirmed that covalently bound radioactivity was represented by fatty acids.     

Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.     

Homology of variable major protein genes between Borrelia hermsii and Borrelia miyamotoi

Abstract Antigenic variation has been studied in detail for the etiological agent of relapsing fever, Borrelia hermsii . The variable major proteins (vmps) are found at its cell surface, enabling it to avoid the host's immune response. We have cloned and sequenced the vmp -gene ( vmp )-like sequences from the Borrelia miyamotoi strains HT31 and FR64b and the deduced amino acid sequences were compared with the published vmp proteins vmp3, vmp24, and vmp33 of B. hermsii . The sequences were aligned and revealed pairwise sequence identities ranging from 45 to 51%, and differences were scattered throughout the sequences. Southern hybridization using the cloned vmp -like sequence of strain HT31 as a probe suggested that the vmp homologues reside on the linear plasmids of B. miyamotoi . The probe hybridized weakly with B. hermsii linear plasmids and restriction digests. These results suggest that B. miyamotoi has sequences resembling the vmp genes in B. hermsii .     

T Cell Response to Borrelia garinii,Borrelia afzelii,and Borrelia japonica in Various Congenic Mouse Strains

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross-reactivity between Borrelia antigens was observed, but there was no cross-reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed-type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4⁺8^? and Ia^? T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8⁺ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.     

Characterization and identification of Borrelia isolates as Borrelia valaisiana in Taiwan and Kinmen Islands

Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.     

Whole-genome sequences of two Borrelia afzelii and two Borrelia garinii Lyme disease agent isolates

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.     

Bacteriolytic activity of selected vertebrate sera for Borrelia burgdorferi sensu stricto and Borrelia bissettii

An in vitro assay to evaluate the bacteriolytic activity of the complement pathway was applied to 2 strains of Borrelia bissettii, CO501 and DN127, and compared with that of B. burgdorferi sensu stricto B31. Sera from mule deer (Odocoileus hemionus) and the Western Fence lizard (Sceloporus occidentalis) were completely borreliacidal for B. burgdorferi and for both strains of B. bissettii. Serum from Bobwhite quail (Colinus virginianus) was nonlytic for B. burgdorferi and partially lytic for B. bissettii strains, CO-501 and DN127. Serum from a New Zealand White rabbit (Oryctolagus cuniculus) was partially lytic for all 3 strains of Borrelia, whereas serum from white-footed mice (Peromyscus leucopus) were nonlytic for all 3 Borrelia strains. The spectrum of complement sensitivity of B. bissettii appears to be similar to that of European B. afzelii in that tested rodent serum is not lytic to these 2 genospecies. Interestingly, both B. bissettii and B. afzelii have been found to be closely associated with rodents. Complement sensitivity demonstrated in these experiments may suggest and possibly predict specific reservoir-host associations.     

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.     

Ultrastructure of Treponema microdentium and Borrelia vincentii

Bladen, Howard A. (National Institute of Dental Health, Bethesda, Md.), and Edward G. Hampp. Ultrastructure of Treponema microdentium and Borrelia vincentii. J. Bacteriol. 87:1180-1191.-A small oral Treponema (FM) and Borrelia vincentii (N9) were harvested after 3 to 7 days of incubation and either embedded in Vestopal W or negatively stained with phosphotungstate. The protoplasmic cylinders of both strains were identical except for size, and had a triple-structured cell wall as well as intracellular concentric laminations. Protoplasmic cylinders of both strains were enclosed in a cell envelope which appeared amorphous in negatively stained preparations, but which had a triple-structured wall when viewed in thin sections. The cell envelope of strain FM also acted as an envelope for the terminal filament; no filament envelope was evident in strain N9. Large structures which contained variable numbers of organisms and which were representative of spirochetal granules were observed. Protoplasmic cylinders contained within such granules frequently were devoid of cell envelopes. The axial filament consisted of several individual fibers which usually terminated in small end knobs. Occasionally, a fiber of the axial filament became a fiber of the terminal filament. Fibers of the terminal filament originated in end knobs similar to, but separate from, those to which the axial filament was attached. A periodicity of 60 A was occasionally observed in the terminal filament envelope of strain FM. A microperiodicity of approximately 20 A was also observed. The fibers of the terminal filament of strain N9 were composed of a large number of fibrils approximately 15 A wide. The periodicity and fibrillar structure of the terminal filament is discussed with reference to proposed models of bacterial flagella suggested by X-ray diffraction data.     

Preservation of Borrelia kansas and Plasmodium berghei

[This corrects the article on p. 224 in vol. 20.].     

Preservation of Borrelia kansas and Plasmodium berghei

Borrelia kansas and Plasmodium berghei have been stored after slow freezing in thioglycollate-glycerol medium for a 6-month period. During this time, 75% or more of the Borrelia remained motile, many intracellular malarial parasites exhibited amoeboid movement, and the growth pattern of both organisms in mice remained unchanged.     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species

Abstract We have cloned and sequenced the flagellin gene from Borrelia miyamotoi strain HT31 and compared it with previously published flagellin sequences. Sequence similarity analysis demonstrated that strain HT31 is phylogenetically distant from the three species of Lyme disease borreliae and is deeply branched into the relapsing fever borrelia cluster. The result was in full agreement with the classification of Borrelia strains using 16S rRNA sequences. This finding indicates that a phylogenetic analysis using flagellin gene sequences might be useful for classification of Borrelia strains.     

Chemotaxis in Borrelia burgdorferi

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.