RESUMEN
Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent, Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. The glpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed the glpQ gene from B. recurrentis and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells of B. recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence.
Asunto(s)
Proteínas Bacterianas/genética , Infecciones por Borrelia/diagnóstico , Borrelia/clasificación , Hidrolasas Diéster Fosfóricas/genética , Phthiraptera/microbiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/análisis , Borrelia/genética , Borrelia/aislamiento & purificación , Infecciones por Borrelia/sangre , Clonación Molecular , Convalecencia , Cartilla de ADN , ADN Bacteriano/genética , Etiopía , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Insectos Vectores , Hidrolasas Diéster Fosfóricas/análisis , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Pruebas SerológicasRESUMEN
Borrelia hermsii, an agent of tick-borne relapsing fever, was found to contain multiple circular plasmids approximately 30 kb in size. Sequencing of a DNA library constructed from circular plasmid fragments enabled assembly of a composite DNA sequence that is homologous to the cp32 plasmid family of the Lyme disease spirochete, B. burgdorferi. Analysis of another relapsing fever bacterium, B. parkeri, indicated that it contains linear homologs of the B. hermsii and B. burgdorferi cp32 plasmids. The B. hermsii cp32 plasmids encode homologs of the B. burgdorferi Mlp and Bdr antigenic proteins and BlyA/BlyB putative hemolysins, but homologs of B. burgdorferi erp genes were absent. Immunoblot analyses demonstrated that relapsing fever patients produced antibodies to Mlp proteins, indicating that those proteins are synthesized by the spirochetes during human infection. Conservation of cp32-encoded genes in different Borrelia species suggests that their protein products serve functions essential to both relapsing fever and Lyme disease spirochetes. Relapsing fever borreliae replicate to high levels in the blood of infected animals, permitting direct detection and possible functional studies of Mlp, Bdr, BlyA/BlyB, and other cp32-encoded proteins in vivo.
Asunto(s)
Antígenos Bacterianos/genética , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/inmunología , Borrelia burgdorferi , Borrelia/genética , Borrelia/inmunología , Plásmidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sondas de ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana. DNA from R. peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16S ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes. Partial 16S rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R. By using Gimenez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected. The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues. Attempts to cultivate R. peacockii were unsuccessful.
Asunto(s)
Rickettsia/clasificación , Rickettsia/genética , Garrapatas/microbiología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Montana , Óvulo/microbiología , Óvulo/ultraestructura , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Rickettsia/aislamiento & purificación , Especificidad de la Especie , Garrapatas/ultraestructuraRESUMEN
Tick-borne relapsing fever is caused by numerous Borrelia species maintained in nature by Ornithodoros tick-mammal cycles. Serological confirmation is based on either an immunofluorescence assay or an enzyme-linked immunosorbent assay using whole cells or sonicated Borrelia hermsii as the antigen. However, antigenic variability of this bacterium's outer surface proteins and antigens shared with the Lyme disease spirochete (B. burgdorferi), may cause both false-negative and false-positive results when testing sera of patients suspected to have either relapsing fever or Lyme disease. To develop a specific serological test for relapsing fever, we created a genomic DNA library of B. hermsii, screened transformed Escherichia coli cells for immunoreactivity with high-titered (> or = 1:2,048) human anti-B. hermsii antiserum, and selected an immunoreactive clone (pSPR75) expressing a 39-kDa protein. DNA sequencing, subcloning, and serum adsorption experiments identified the immunoreactive protein as a homolog of GlpQ, a glycerophosphodiester phosphodiesterase identified previously in E. coli, Haemophilus influenzae, and Bacillus subtilis. Serum samples from humans and mice infected with B. hermsii or other species of relapsing fever spirochetes contained antibodies recognizing GlpQ, whereas serum samples from Lyme disease and syphilis patients were nonreactive. Serologic tests based on this antigen will identify people exposed previously to relapsing fever spirochetes and help clarify the distribution of relapsing fever and Lyme disease in situations in which the occurrence of their causative agents is uncertain.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Borrelia/inmunología , Enfermedad de Lyme/diagnóstico , Hidrolasas Diéster Fosfóricas/inmunología , Fiebre Recurrente/diagnóstico , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Borrelia/genética , ADN Bacteriano/análisis , Diagnóstico Diferencial , Humanos , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Ratones , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/genética , Fiebre Recurrente/inmunología , Fiebre Recurrente/microbiología , Alineación de Secuencia , Pruebas SerológicasRESUMEN
The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyme disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Grupo Borrelia Burgdorferi/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Grupo Borrelia Burgdorferi/genética , ADN Bacteriano/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We used the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) rickettsial typing system of Regnery and others to rapidly identify rickettsiae in naturally infected ticks. Unlike previously described methods, our PCR assays type rickettsiae directly from tick tissues without first isolating the organisms. We collected 226 adult Dermacentor andersoni ticks in the Bitterroot Mountains of western Montana and analyzed them for possible rickettsial infection by hemolymph test using the Gimenez stain. Thirteen (5.8%) of these ticks were positive by hemolymph test and selected for further analysis using the above PCR/RFLP typing system. The PCR assays performed using the first primer set (RpCS) resulted in amplification of fragments of the predicted size from nine of the 13 hemolymph test-positive tick samples. Only four of these nine tick samples were also positive in similar PCR assays performed with a second primer set (Rr190) that is presumed to be spotted fever group specific. The RFLP analyses of material amplified from these four ticks indicated they were infected with Rickettsia rickettsii (one sample) and R. rhipicephali (three samples). The PCR/RFLP analyses of the five PCR-positive tick samples that were positive only in assays performed with the RpCS primer set indicated that these ticks were infected with R. bellii. The remaining four of 13 hemolymph test-positive tick samples gave negative PCR results with both the RpCS and Rr190 primer sets. Infected hemocytes from these PCR-negative ticks contained organisms of distinctive bacillary morphology that appeared similar to those described previously as long forms, and it is possible that these organisms belong to a genus other than Rickettsia. We also examined established laboratory isolates of tick-borne rickettsiae from different regions of North America to determine whether this typing system produces consistent results. Multiple isolates of R. montana (nine isolates), R. bellii (five isolates), R. rickettsii (Hlp-like) (four isolates), and R. canada (two isolates) were tested and no significant variations in PCR/RFLP patterns were observed between members of the same serotypes. However, among the five isolates of R. rhipicephali tested, two slightly different RFLP patterns were noted. Our results suggest that this PCR/RFLP typing scheme has wide applicability for identifying rickettsiae directly from D. andersoni or D. variabilis tick tissues.
Asunto(s)
Vectores Arácnidos/microbiología , ADN Bacteriano/análisis , Dermacentor/microbiología , Polimorfismo de Longitud del Fragmento de Restricción , Rickettsia/clasificación , Animales , Composición de Base , Cartilla de ADN/química , Hemolinfa/microbiología , Ontario , Reacción en Cadena de la Polimerasa , Rickettsia/genética , Serotipificación , Estados UnidosRESUMEN
Previous studies describing the occurrence and molecular characteristics of Lyme disease spirochetes, Borrelia burgdorferi, from California have been restricted primarily to isolates obtained from the north coastal region of this large and ecologically diverse state. Our objective was to look for and examine B. burdorferi organisms isolated from Ixodes pacificus ticks collected from numerous regions spanning most parts of California where this tick is found. Thirty-one isolates of B. burgdorferi were examined from individual or pooled I. pacificus ticks collected from 25 counties throughout the state. One isolate was obtained from ticks collected at Wawona Campground in Yosemite National Park, documenting the occurrence of the Lyme disease spirochete in an area of intensive human recreational use. One isolate from an Ixodes neotomae tick from an additional county was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, agarose gel electrophoresis, Southern blot analysis, and the polymerase chain reaction were used to examine the molecular and genetic determinants of these uncloned, low-passage-number isolates. All of the isolates were identified as B. burgdorferi by their protein profiles and reactivities with monoclonal and polyclonal antibodies, and all the isolates were typed by the polymerase chain reaction as North American-type spirochetes (B. burgdorferi sensu stricto). Although products of the ospAB locus were identified in protein analyses in all of the isolates, several isolates contained deleted forms of this locus that would result in the expression of chimeric OspA-OspB proteins. The analysis of OspC demonstrated that this protein was widely conserved among the isolates but was also quite variable in its molecular mass and the amount of it that was expressed.
Asunto(s)
Grupo Borrelia Burgdorferi/aislamiento & purificación , Garrapatas/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Vectores Arácnidos/microbiología , Secuencia de Bases , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/inmunología , California , ADN Bacteriano/genética , Femenino , Genes Bacterianos , Humanos , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/transmisión , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Peromyscus , Plásmidos/genética , Plásmidos/aislamiento & purificación , Reacción en Cadena de la PolimerasaRESUMEN
Borrelia hermsii causes a relapsing fever in humans and is one of several species of tick-borne spirochetes known to occur in the western United States. Spirochetes observed in the peripheral blood of patients acutely ill have been presumptively identified in the past by the geographic location of exposure and the probable species of tick vector. We describe a monoclonal antibody (H9826) that bound to the flagellar protein of B. hermsii but not to those of any of the other species tested, which included B. parkeri, B. turicatae, B. coriaceae, B. anserina, B. burgdorferi, and Leptospira interrogans serovar ballum. This antibody bound efficiently to B. hermsii in an indirect immunofluorescence assay and was used to rapidly detect and identify this spirochete in the peripheral blood of experimentally infected mice and in the central ganglia of Ornithodoros hermsi ticks. H9826 can rapidly confirm the identification of B. hermsii to increase our understanding concerning the geographic distribution, vector specificity, and epidemiological significance of this zoonotic human pathogen.
Asunto(s)
Anticuerpos Monoclonales , Borrelia/inmunología , Fiebre Recurrente/microbiología , Anticuerpos Antibacterianos , Borrelia/aislamiento & purificación , Borrelia/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Inmunoelectrónica , Fiebre Recurrente/diagnóstico , Especificidad de la EspecieRESUMEN
Five serovars of Leptospira interrogans, Leptospira biflexa, Leptonema illini, and Rickettsia rickettsii were examined and found not to contain the 39-kDa antigen (P39) of Borrelia burgdorferi, the Lyme disease spirochete. The specificity of this antigen and its reactivity with human Lyme disease sera should exclude the possibility of false-positive serum samples from patients having had either leptospirosis or Rocky Mountain spotted fever, as well as tick-borne relapsing fever and syphilis, as reported previously (W.J. Simpson, M. E. Schrumpf, and T. G. Schwan, J. Clin. Microbiol. 28:1329-1337, 1990).
Asunto(s)
Antígenos Bacterianos/análisis , Grupo Borrelia Burgdorferi/inmunología , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Reacciones Cruzadas , Reacciones Falso Positivas , Humanos , Leptospira/inmunología , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/inmunología , Peso Molecular , Rickettsia rickettsii/inmunología , Pruebas Serológicas , Especificidad de la EspecieRESUMEN
Borrelia burgdorferi is the causative agent of Lyme disease, a tick-borne spirochetosis with a worldwide prevalence. To assist the categorization and typing of fresh isolates from global foci, we have identified a unique species-specific periplasmic protein (P22-A) conserved among all North American and European isolates examined. The gene encoding this antigen was cloned, and the recombinant was used to screen serum collected from experimentally infected animals. Although antibodies were detected in all infected animals at 21 days after inoculation with live, low-passage spirochetes, the response was stronger in other animals that were inoculated with inactivated and lysed bacteria. This result, along with the immune electron microscopy data, suggests P22-A is concentrated in the periplasmic space. The P22-A antigens exhibited size heterogeneity among different isolates, ranging between 20 and 23 kDa, but as a group the P22-A antigens appeared to retain antigenic homogeneity. Thus, P22-A can serve as a structural marker for characterizing new isolates of B. burgdorferi and may prove useful in future serological assays with a mixture of B. burgdorferi-specific antigens.
Asunto(s)
Proteínas Bacterianas/inmunología , Grupo Borrelia Burgdorferi/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/genética , Biomarcadores , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/ultraestructura , Clonación Molecular , ADN Bacteriano/genética , Inmunoquímica , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/inmunología , Ratones , Polimorfismo GenéticoRESUMEN
Adult laboratory mice, Mus musculus, were shown to be suitable experimental animals for studying infectivity, persistent infection, and in vivo antigenic changes of Borrelia burgdorferi. Sixteen mice were inoculated intraperitoneally with a low-passage culture of an uncloned strain of B. burgdorferi and 16 months later spirochetes were reisolated from the urinary bladder of 15 (94%) of the mice. Spirochetes recovered from the urinary bladder of one persistently infected mouse were tested for infectivity and found to be infectious when passaged into four laboratory mice. Western blot analysis of immune serum from each of the persistently infected mice demonstrated that spirochetes used to infect the mice reacted differently when compared with the spirochetes subsequently reisolated from the mice, demonstrating for the first time that changes in antigenic reactivity had occurred in the spirochete populations during persistent infection.
Asunto(s)
Antígenos Bacterianos/inmunología , Grupo Borrelia Burgdorferi/inmunología , Borrelia burgdorferi , Enfermedad de Lyme/inmunología , Animales , Variación Antigénica , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ratones , Ratones EndogámicosRESUMEN
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.
Asunto(s)
Antígenos Bacterianos , Grupo Borrelia Burgdorferi/inmunología , Enfermedad de Lyme/diagnóstico , Animales , Anticuerpos Antibacterianos/biosíntesis , Biomarcadores , Grupo Borrelia Burgdorferi/crecimiento & desarrollo , Grupo Borrelia Burgdorferi/patogenicidad , Femenino , Flagelina/inmunología , Enfermedad de Lyme/inmunología , Ratones , Ratones Endogámicos BALB C , PeromyscusRESUMEN
Borrelia burgdorferi is the causative agent of Lyme borreliosis, a spirochetal illness with a variety of acute clinical manifestations that may lead to debilitating neurological and arthritic complications. Diagnosis is difficult because symptoms mimic a variety of unrelated clinical conditions, spirochetes cannot always be isolated from infected patients, and current serological tests are frequently inconclusive because of the presence of cross-reacting non-B. burgdorferi antibodies. To identify antigens specific to B. burgdorferi that could be used in the serodiagnosis of Lyme borreliosis, we screened a Borrelia DNA expression library in Escherichia coli for antigens reactive with human Lyme borreliosis sera. One clone carried a 6.3-kilobase EcoRI chromosomal fragment (pSPR33), which encoded two species-specific antigens with molecular masses of 28 (P28) and 39 (P39) kilodaltons (kDa). These two antigens were immunologically distinct from OspA, OspB, and the 41-kDa flagellin. Ninety-four serum specimens from patients having Lyme borreliosis were tested for reactivity with P39. All of 33 the serum specimens with immunofluorescence assay titers of greater than or equal to 1:256, 13 of 17 serum specimens with titers of 1:128, and 14 of 44 serum specimens with titers of less than or equal to 1:64 reacted with P39. Notably, many sera reactive to P39 did not appear to react with the 41-kDa flagellin. Therefore, antibody to P39 could be mistaken for antibody to the 41-kDa flagellin in tests of human sera by Western blot (immunoblot). Twenty-five control serum specimens, which included sera from syphilitic, relapsing fever, and amyotrophic lateral sclerosis patients as well as from 10 normal individuals, did not react to P39. Our data suggest that P39 may be a useful antigen for the serological confirmation of Lyme borreliosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Borrelia/inmunología , Grupo Borrelia Burgdorferi/inmunología , Animales , Western Blotting , Grupo Borrelia Burgdorferi/genética , Reacciones Cruzadas , ADN Bacteriano/análisis , Humanos , Peso Molecular , Conejos , Sífilis/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Grupo Borrelia Burgdorferi/inmunología , Borrelia burgdorferi , Enfermedad de Lyme/inmunología , Peromyscus/inmunología , Animales , Antígenos Bacterianos/inmunología , Western Blotting , Inmunoglobulina G/biosíntesis , Isotipos de Inmunoglobulinas/biosíntesis , Inmunoglobulina M/biosíntesis , Peso Molecular , Factores de TiempoRESUMEN
Fragments of plasmid DNA from Borrelia burgdorferi and B. hermsii were cloned and tested for specificity as hybridization probes to identify these two species of pathogenic spirochetes. Three fragments from the 49-kilobase-pair linear plasmid of B. burgdorferi were tested: a 500-base-pair (bp) HindIII fragment (probe 49A), a 445-bp PstI-HindIII fragment (probe 3G), and a 320-bp HindIII fragment (probe 16H). When hybridized to purified DNA or whole spirochetes, all of the probes distinguished B. burgdorferi from the other species examined, including B. hermsii, B. parkeri, B. turicatae, B. coriaceae, B. crocidurae, and B. anserina. Probe 49A was the most useful, however, hybridizing with all strains of B. burgdorferi originating from both North America and Europe while not cross-hybridizing with B. hermsii. A 790-bp HindIII fragment of B. hermsii DNA hybridized with DNA and whole spirochetes of this species and also with B. parkeri, confirming the close relatedness of these two species. These probes provide a new method of identifying these Borrelia species once the organisms have been grown in culture.
Asunto(s)
Borrelia/aislamiento & purificación , Sondas de ADN , ADN Bacteriano/análisis , Animales , Southern Blotting , Borrelia/genética , Reacciones Cruzadas , Humanos , Hibridación de Ácido Nucleico , Plásmidos , GarrapatasRESUMEN
White-footed mice, Peromyscus leucopus, were experimentally infected in the laboratory with Borrelia burgdorferi, the causative agent of Lyme disease. After mice were infected by intraperitoneal or subcutaneous inoculation or by tick bite, attempts were made to culture spirochetes from the urinary bladder, spleen, kidney, blood, and urine. Spirochetes were most frequently isolated from the bladder (94%), followed by the kidney (75%), spleen (61%), and blood (13%). No spirochetes were isolated from the urine. Tissue sectioning and immunofluorescence staining of the urinary bladder demonstrated spirochetes within the bladder wall. The results demonstrate that cultivation of the urinary bladder is very effective at isolating B. burgdorferi from experimentally infected white-footed mice and that culturing this organ may be productive when surveying wild rodents for infection with this spirochete.
Asunto(s)
Borrelia/aislamiento & purificación , Enfermedad de Lyme/veterinaria , Peromyscus , Enfermedades de los Roedores/microbiología , Vejiga Urinaria/microbiología , Animales , Borrelia/crecimiento & desarrollo , Borrelia/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente , Riñón/microbiología , Enfermedad de Lyme/microbiología , Masculino , Microscopía Electrónica de Rastreo , Bazo/microbiología , Vejiga Urinaria/ultraestructuraRESUMEN
Forty-five isolates of Borrelia burgdorferi, the etiologic agent of Lyme disease, were examined with monoclonal antibodies directed against the surface proteins, OspA and OspB. Eighteen of 21 strains of European origin could be distinguished from 24 North American strains on the basis of their reactivities with two antibodies, one specific for an OspA protein and one specific for an OspB protein. Differences in antibody reactivities were associated with differences in the electrophoretic migrations of OspA and OspB proteins. Thus, Lyme disease borreliae are polymorphic with respect to their major surface proteins. The demonstrated distinctions between the majorities of European and North American strains may have relevance for both epidemiologic and pathogenic studies of Lyme disease.
Asunto(s)
Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Borrelia/inmunología , Proteínas de la Membrana/análisis , Polimorfismo Genético , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Borrelia/genética , Electroforesis en Gel de Poliacrilamida , Epítopos/análisis , Europa (Continente) , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , América del NorteRESUMEN
In immunofluorescence assays monoclonal antibody H9724 recognized eight species of the spirochetal genus Borrelia but not representatives of the genera Treponema, Leptospira, and Spirochaeta. We examined the reactivity of H9724 against subcellular components of Borrelia hermsii, an agent of relapsing fever, and B. burgdorferi, the cause of Lyme disease. H9724 bound to isolated periplasmic flagella of the two borreliae. In Western blots the antibody reacted with the predominant protein in flagellar preparations from B. hermsii and B. burgdorferi; the apparent molecular weights of these flagellins were 39,000 and 41,000, respectively.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Borrelia/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Borrelia/ultraestructura , Flagelos/inmunología , Técnica del Anticuerpo Fluorescente , Oro , Microscopía Electrónica , Peso MolecularRESUMEN
Most of the isolates of Bordetella bronchiseptica obtained by this laboratory possessed a characteristic colonial morphology when grown on Bordet- Gengou agar (BGA) at 37 degrees C. The colonies appeared domed (Dom+) with a smooth colonial surface (Scs+) and a clear zone of hemolysis ( Hly +). From these Dom+ Scs+ Hly + BGA colony types arose flat (Dom-), smooth colonial surface (Scs+) and nonhemolytic ( Hly -) variants at frequencies of 10(-2) to 10(-3). Isogenic pairs of Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA phenotype variants (BGA-PVs) were picked from 11 strains of B. bronchiseptica, and their whole cell lysates were compared with each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Characteristic SDS-PAGE profiles were observed for each of the Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs with regard to (i) surface-exposed proteins, based on autoradiographs of 125I- Iodogen -labeled organisms, (ii) polypeptide differences, based on gels stained with Coomassie brilliant blue R-250, and (iii) lipopolysaccharide differences based on gels stained with silver after oxidation with periodic acid. SDS-PAGE profiles were then used to monitor the phenotypes expressed by Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs transferred and grown on brucella agar, Trypticase soy agar, and nutrient agar. When grown on non-BGA media, the Dom+ Scs+ Hly + BGA-PVs from six of eight strains showed SDS-PAGE profiles identical to those of Dom- Scs+ Hly - BGA-PVs. This phenotypic change was reversible even after 15 subcultures on the non-BGA media, since Dom+ Scs+ Hly + organisms passed back onto BGA expressed both Dom+ Scs+ Hly + colonial morphology and Dom+ Scs+ Hly + SDS-PAGE profiles. The influence of cultural conditions on maintenance of virulence is discussed.
Asunto(s)
Técnicas Bacteriológicas , Bordetella/inmunología , Animales , Proteínas Bacterianas/análisis , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Cobayas , FenotipoRESUMEN
Unsupplemented nutrient agar (NA) was used to select spontaneous phenotype variants (PVs) of Bordetella pertussis Tohama I and 3779 which, by their growth on NA, could possibly be considered equivalent to phase IV in the system of Leslie and Gardner (P.H. Leslie and A.D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1953). NA growers (Gna+) were selected from the flat, nonhemolytic, non-NA grower (Dom- Hly- Gna-) PV of both strains at a rate of between 10(-7) to 10(-8) per cell per generation. When cultured on Bordet-Gengou agar (BGA), more than one colony type was observed in strain 3779; these all retained the Gna+ characteristic during 10 to 30 passages on BGA. Analysis of 125I-surface-labeled whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no major changes between the Dom- Hly- Gna+ PV and their Dom- Hly- Gna- PV parents in polypeptide profile (by Coomassie stain), in surface exposure of proteins (by autoradiography), or in lipopolysaccharide profile (by silver stain). Increased resistance to oleic acid, tetracycline, erythromycin, rifampin, and penicillin G, however, was characteristic for the Dom- Hly- Gna+ PV. Five phase IV strains and a phase III B. pertussis strain had similar antibiotic and oleic acid sensitivity profiles as the Dom- Hly- Gna+ isolates and plated with similar efficiency on NA, despite heterogeneity in BGA colonial morphology and lipopolysaccharide profile.