Chromosomal diversity in Hypostomus (Siluriformes, Loricariidae) with emphasis on physical mapping of 18S and 5S rDNA sites.

We examined chromosomes of three species of the genus Hypostomus, in order to contribute to the understanding of the karyotype evolution of this group. Specimens of H. ancistroides and H. nigromaculatus displayed differences in karyotype formulas, distribution and location of heterochromatin and nucleolus organizer regions when compared to other populations of the same species. We made the first cytogenetic characterization of H. tapijara, an endemic species in the Ribeira de Iguape River. These specimens had 2n = 66 chromosomes, while H. ancistroides showed 2n = 68 and H. nigromaculatus 2n = 76 chromosomes. Physical mapping of 18S and 5S rDNA sites of the three species showed simple, multiple and syntenic clusters. Synteny of ribosomal sites was found in H. ancistroides and H. tapijara, and an interspersed pattern between these sites in all chromosomes bearing the synteny was observed. We conclude that the genus Hypostomus has a high chromosome complexity that is accompanied by great morphological variation. It is evident that this group comprises an interesting model for understanding the chromosome evolution of Neotropical ichthyofauna.

A new technique for obtaining mitotic chromosome spreads from fishes in the field.

This study presents an adaptation of current methodologies for preparing mitotic chromosomes from fishes, optimized for use in the field. The high-quality preparations obtained using this modified methodology is suitable for subsequent chromosomal analysis. Importantly, this method is particularly useful when specimen collection sites are far from research laboratories or when researchers are working with highly sensitive species that do not survive long outside of their natural habitats.

Comparative cytogenetics of giant trahiras Hoplias aimara and H. intermedius (Characiformes, Erythrinidae): chromosomal characteristics of minor and major ribosomal DNA and cross-species repetitive centromeric sequences mapping differ among morphologically identical karyotypes.

Karyotype and cytogenetic characteristics of 2 species of giant trahiras, Hopliasintermedius, São Francisco river basin, and Hopliasaimara, Arinos river (Amazon basin), were examined by conventional (C-banding, Ag-NOR, DAPI/CMA(3) double-staining) and fluorescent in situ hybridization (FISH) with 5S, 18S rDNA probes and cross-species Cot-1 DNA probing. Both species invariably had diploid chromosome number 2n = 50 and identical karyotypes composed of 10 pairs of metacentric and 15 pairs of submetacentric chromosomes. On the other hand, staining with base-specific fluorochromes (CMA(3), DAPI) and FISH mapping of repetitive DNA sequences showed extensive interspecific differences: while the genome of H. aimara had one submetacentric pair bearing CMA(3)-positive (DAPI-negative) sites, that of H. intermedius had 4 such pairs; while FISH with a 5S rDNA probe showed one (likely homologous) signal-bearing pair, that with 18S rDNA displayed one signal-bearing pair in H. intermedius and 2 such pairs in H. aimara. Cross-species FISH probing with Cot-1 DNA prepared from total DNA of both species showed no signals of Cot-1 DNA from H. aimara on chromosomes of H. intermedius but reciprocally (Cot-1 DNA from H. intermedius on chromosomes of H. aimara) displayed signals on at least 4 chromosome pairs. Present findings indicate (i) different composition of repetitive sequences around centromeres, (ii) different NOR phenotypes and (iii) distinct taxonomic status of both giant trahira species.

Acquired resistance to Borrelia burgdorferi infection in the rabbit. Comparison between outer surface protein A vaccine- and infection-derived immunity.

Intradermal inoculation of the rabbit with Borrelia burgdorferi, sensu lato, results in the consistent development of erythema migrans (EM), dermal infection, and visceral dissemination of the spirochete. Within 5 mo, EM as well as dermal and visceral infection are cleared and the animals exhibit immunity to reinfection. This study compares infection-derived immunity with acquired resistance resulting from the administration of a lipidated recombinant outer surface protein A (OspA) vaccine presently undergoing human trial. 4 of 11 OspA vaccinated rabbits, challenged intradermally at each of 10 sites with 10(5) low passage B. burgdorferi, developed EM as well as dermal and disseminated infection. After identical challenge, 2 of the 11 infection-immune rabbits developed a dermal infection, but not EM or disseminated infection. Further, ELISA anti-OspA titers did not correlate with the status of immunity for either OspA vaccinated or infection-immune rabbits. Prechallenge ELISA anti-OspA titers were relatively low in the infection-immune group. This study demonstrates that a state of partial immunity to experimental Lyme disease may result that could potentially mask infection. Further, our data strongly suggest that immunogen(s) other than OspA is/are responsible for stimulating acquired resistance in the infection-immune rabbit.

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Rabbit model of Lyme borreliosis: erythema migrans, infection-derived immunity, and identification of Borrelia burgdorferi proteins associated with virulence and protective immunity.

Erythema migrans (EM), persistent skin infection, and visceral dissemination can be induced reproducibly in the adult male New Zealand White rabbit by intradermal injection of as few as 10(3) Borrelia burgdorferi. EM was found to persist for 7 +/- 3 d. Skin culture positivity (infection) cleared within a mean of 6.7 +/- 1.4 wk after infection and similarly visceral infection was not demonstrated after 8 wk; infection-derived immunity to intradermal challenge was evident 5 mo after initial infection. The extent of the protection against EM and dermal infection induced by untreated infection was directly related to the extent of prior in vitro passage of the B31 strain. Initial infection with as few as 4 x 10(3) B31 passage 4 induced complete protection against EM and skin infection upon subsequent challenge with 4 x 10(7) B31, passage 4. Initial infection with B31 passage 27 led to partial protection against EM along with complete protection against skin infection. Initial infection with passage 47 led to partial protection against EM, but conferred no protection against skin infection. Using serum from rabbits fully immune to reinfection, we defined a set of B. burgdorferi proteins present in virulent B31, but absent in the avirulent American Type Culture Collection B31 strain, termed "va" for virulent strain associated. The va proteins of B31 passages 1, 27, and 47 differed strikingly, thus raising the possibility that these changes may relate in a causal way to the differences in induction of protective immunity observed.

Karyotypic variation of Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae) along the Iguazu river basin.

The Iguazu river is a tributary of the left margin of the Paraná river, isolated from this basin about 22 million years ago with the appearance of the Iguazu Falls. The Iguazu river is characterized by high endemism due to two factors: its rugged topography and the old isolation caused by formation of the Iguazu Falls. This study analyzed cytogenetically a population of Glanidium ribeiroi collected in a region at the final stretch of this basin, by Giemsa staining, C-banding, impregnation by silver nitrate, and FISH with probes of 5S rDNA, 18S rDNA, telomeric sequence [TTAGGG]n, and [GATA]n repeats. The diploid number was equal to 58 chromosomes. The heterochromatin was present in the terminal region of almost all chromosomes. The Ag-NORs were simple and presented interstitially on the short arm of the submetacentric pair 14, which was confirmed by FISH with 18S rDNA probe. The 5S rDNA-FISH marked only the submetacentric pair 16 on the long arm in interstitial position. The FISH with [TTAGGG]n probe presented all telomeres labeled as expected, with an absence of Interstitial Telomeric Sequence (ITS). The repetitive [GATA]n sequence was dispersed throughout the genome, with preferential location in the terminal region of all chromosomes. The data obtained are discussed herein with other species of Auchenipteridae, and other previously analyzed populations of G. ribeiroi from the Iguazu river, verifying differences among these populations, which should be mainly related to the rugged topography of this basin.

Montagem de cariótipos de peixes assistida por computador / Computer assisted fish karyotyping

A obtenção de cariótipos de peixes desempenha um papel importante em estudos de citotaxonomia e evolução cromossômica das espécies. No entanto, poucos sistemas semi ou completamente automatizados para a obtenção de cariótipo de peixes estão disponíveis. Este trabalho propõe e avalia uma ferramenta baseada em imagens que auxilie a montagem de cariótipos de peixes. As espécies analisadas foram Hopliasmalabaricus (Bloch, 1794); Hypostomusancistroides (Ihering, 1911) e Parauchenipterusgaleatus (Linnaeus, 1766); popularmente conhecidas como traíra, cascudo e bagre-sapo, respectivamente.Um total de 100 metáfases foi analisado por dois métodos: 1 - geração semiautomática de cariótipo e 2 - geração automática de cariótipo. A avaliação do sistema foi feita por meio da correlação de Pearson, gráficos de diferenças e tabelas de contagens,utilizando-se como referência a média das contagens feitas por quatro usuários. No método 1, quatro usuários realizaram contagens e apresentaram correlação interobservador de r≥ 0.997. O número total de cromossomos identificados pelo método 1 foi 4348 e, para o método 2, foi 4135,o que resultou em uma identificação automática de aproximadamente 95,1% dos cromossomos, resultando em correlação entre os dois métodos de r= 0.93. Conclui-se que a ferramenta pode ser inserida no procedimento de cariotipagem de peixes para acelerar o processo com níveis aceitáveis de exatidão.(AU)

Fish karyotyping plays an important role in studies of cytotaxonomy and chromosomic evolution of species. However, few semi or completely automated fish karyotyping systems are available. This work proposes and evaluates an image-based tool to assist fish karyotyping. The analyzed species were Hopliasmalabaricus (Bloch, 1794), Hypostomusancistroides (Ihering, 1911), and Parauchenipterusgaleatus (Linnaeus, 1766). In Portuguese, these species are commonly referred to as traíra, cascudo, and bagresapo, respectively. A total of 100 metaphases were analyzed through two methods: (1) semi-automatic karyotype generation and (2) automatic karyotype generation. The results were analyzed using Pearson correlation, difference graphs and counting tables. The reference used for the evaluation of the system was the average of the counts made by four experts. In method 1, four users performed counts with interobserver correlation of r≥ 0.997. The total number of chromosomes identified by method 1 was 4348 and method 2 was 4135, excluding false positives, resulting in an automatic identification of approximately 95,1% of the chromosomes, resulting in a correlation between the methods of r= 0.93. The results indicate that the tool can be introduced for fish karyotyping procedures contributing for accelerating the process with acceptable accuracy.(AU)

Propidium iodide for making heterochromatin more evident in the C-banding technique.

The detection of regions of heterochromatin has been the subject of intense investigation. We investigated an adaptation of the commonly used technique by replacing the nonfluorescent dye, Giemsa, by a fluorescent one, propidium iodide. This adaptation produces greater contrast of the heterochromatic bands in metaphase chromosomes and can be especially valuable when the organisms studied possess heterochromatin that is pale and difficult to visualize. We discuss the interactions of these two dyes with DNA and the excitation of the fluorescent dye when irradiated with ultraviolet light.

Karyotypic variation of Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae) along the Iguazu river basin

Abstract The Iguazu river is a tributary of the left margin of the Paraná river, isolated from this basin about 22 million years ago with the appearance of the Iguazu Falls. The Iguazu river is characterized by high endemism due to two factors: its rugged topography and the old isolation caused by formation of the Iguazu Falls. This study analyzed cytogenetically a population of Glanidium ribeiroi collected in a region at the final stretch of this basin, by Giemsa staining, C-banding, impregnation by silver nitrate, and FISH with probes of 5S rDNA, 18S rDNA, telomeric sequence [TTAGGG]n, and [GATA]n repeats. The diploid number was equal to 58 chromosomes. The heterochromatin was present in the terminal region of almost all chromosomes. The Ag-NORs were simple and presented interstitially on the short arm of the submetacentric pair 14, which was confirmed by FISH with 18S rDNA probe. The 5S rDNA-FISH marked only the submetacentric pair 16 on the long arm in interstitial position. The FISH with [TTAGGG]n probe presented all telomeres labeled as expected, with an absence of Interstitial Telomeric Sequence (ITS). The repetitive [GATA]n sequence was dispersed throughout the genome, with preferential location in the terminal region of all chromosomes. The data obtained are discussed herein with other species of Auchenipteridae, and other previously analyzed populations of G. ribeiroi from the Iguazu river, verifying differences among these populations, which should be mainly related to the rugged topography of this basin.

Resumo O rio Iguaçu é um afluente da margem esquerda do rio Paraná, que foi separado desta bacia a aproximadamente 22 milhões de anos com o surgimento das Cataratas do Iguaçu. Esse rio é caracterizado por elevado endemismo, o que se deve a dois fatores: sua acidentada topografia e ao antigo isolamento proporcionado pela formação das cataratas. No presente trabalho foi analisado cromossomicamente uma população de Glanidium ribeiroi coletada em uma região que corresponde ao trecho final desse rio, através de coloração com Giemsa, bandamento-C, impregnação pelo nitrato de prata e FISH com sondas de rDNA 5S, rDNA 18S, sequência telomérica [TTAGGG]n e repetições [GATA]n. O número diploide encontrado foi igual a 58 cromossomos. A heterocromatina se mostrou dispersa na região terminal de quase todos os cromossomos. As Ag-RONs são simples e presentes no braço curto em posição intersticial do par submetacêntrico 14, o que foi confirmado pela FISH com rDNA 18S. O rDNA 5S marcou apenas o par submetacêntrico 16 no braço longo em posição intersticial. A hibridização com sonda [TTAGGG]n revelou todos os telômeros marcados conforme esperado e ausência de Sequência Telomérica Intersticial (ITS). As repetições [GATA]n se apresentaram dispersas no genoma da espécie, com preferencial localização na região terminal de todos os cromossomos. Os dados aqui obtidos são discutidos com os de outras espécies de Auchenipteridae, especialmente de G. ribeiroi anteriormente analisados do rio Iguaçu. Diferenças populacionais são constatadas em decorrência do isolamento geográfico ocasionado pelas inúmeras cachoeiras existentes no curso do rio Iguaçu.

Karyotypic variation of Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae) along the Iguazu river basin / Variação cariotípica de Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae) ao longo da bacia do rio Iguaçu

Abstract The Iguazu river is a tributary of the left margin of the Paraná river, isolated from this basin about 22 million years ago with the appearance of the Iguazu Falls. The Iguazu river is characterized by high endemism due to two factors: its rugged topography and the old isolation caused by formation of the Iguazu Falls. This study analyzed cytogenetically a population of Glanidium ribeiroi collected in a region at the final stretch of this basin, by Giemsa staining, C-banding, impregnation by silver nitrate, and FISH with probes of 5S rDNA, 18S rDNA, telomeric sequence [TTAGGG]n, and [GATA]n repeats. The diploid number was equal to 58 chromosomes. The heterochromatin was present in the terminal region of almost all chromosomes. The Ag-NORs were simple and presented interstitially on the short arm of the submetacentric pair 14, which was confirmed by FISH with 18S rDNA probe. The 5S rDNA-FISH marked only the submetacentric pair 16 on the long arm in interstitial position. The FISH with [TTAGGG]n probe presented all telomeres labeled as expected, with an absence of Interstitial Telomeric Sequence (ITS). The repetitive [GATA]n sequence was dispersed throughout the genome, with preferential location in the terminal region of all chromosomes. The data obtained are discussed herein with other species of Auchenipteridae, and other previously analyzed populations of G. ribeiroi from the Iguazu river, verifying differences among these populations, which should be mainly related to the rugged topography of this basin.

Resumo O rio Iguaçu é um afluente da margem esquerda do rio Paraná, que foi separado desta bacia a aproximadamente 22 milhões de anos com o surgimento das Cataratas do Iguaçu. Esse rio é caracterizado por elevado endemismo, o que se deve a dois fatores: sua acidentada topografia e ao antigo isolamento proporcionado pela formação das cataratas. No presente trabalho foi analisado cromossomicamente uma população de Glanidium ribeiroi coletada em uma região que corresponde ao trecho final desse rio, através de coloração com Giemsa, bandamento-C, impregnação pelo nitrato de prata e FISH com sondas de rDNA 5S, rDNA 18S, sequência telomérica [TTAGGG]n e repetições [GATA]n. O número diploide encontrado foi igual a 58 cromossomos. A heterocromatina se mostrou dispersa na região terminal de quase todos os cromossomos. As Ag-RONs são simples e presentes no braço curto em posição intersticial do par submetacêntrico 14, o que foi confirmado pela FISH com rDNA 18S. O rDNA 5S marcou apenas o par submetacêntrico 16 no braço longo em posição intersticial. A hibridização com sonda [TTAGGG]n revelou todos os telômeros marcados conforme esperado e ausência de Sequência Telomérica Intersticial (ITS). As repetições [GATA]n se apresentaram dispersas no genoma da espécie, com preferencial localização na região terminal de todos os cromossomos. Os dados aqui obtidos são discutidos com os de outras espécies de Auchenipteridae, especialmente de G. ribeiroi anteriormente analisados do rio Iguaçu. Diferenças populacionais são constatadas em decorrência do isolamento geográfico ocasionado pelas inúmeras cachoeiras existentes no curso do rio Iguaçu.

Humoral immunity in experimental syphilis: the demonstration of IgG as a treponemicidal factor in immune rabbit serum.

The neutralizing activity present in immune rabbit serum (IRS) against virulent Treponema pallidum was shown to be mediated by IgG and complement. IgG was isolated and purified from both IRS and nonimmune rabbit serum (NRS) by the use of an affinity system in which staphylococcal protein A was conjugated to Sepharose 4B. The purity of the isolated IgG fractions was demonstrated by both immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Fractions of IgG were tested for specific neutralizing activity as measured by an in vitro-in vivo neutralization test. Lesions failed to develop at 80% of the sites inoculated with treponemal suspensions containing IgG from IRS in the presence of unheated NRS as a source of complement; delayed atypical lesions were observed at the remaining sites. In contrast, typical lesions developed at all sites inoculated with suspensions containing IgG from IRS in the presence of heated NRS. They were significantly delayed, however, as compared with lesion development at control sites inoculated with suspensions containing IgG from NRS. These results provide the first direct evidence for an IgG complement-mediated treponemicidal mechanism operative in immune serum from rabbits with latent syphilis.

Surface antigens of the syphilis spirochete and their potential as virulence determinants.

A unique physical feature of Treponema pallidum, the venereally transmitted agent of human syphilis, is that its outer membrane contains 100-fold less membrane-spanning protein than the outer membranes of typical gram-negative bacteria, a property that has been related to the chronicity of syphilitic infection. These membrane-spanning T. pallidum rare outer membrane proteins, termed TROMPs, represent potential surface-exposed virulence determinants and targets of host immunity. Only recently has the outer membrane of T. pallidum been isolated and its constituent proteins identified. Five proteins of molecular mass 17-, 28-, 31-, 45-, and 65-kDa were outer membrane associated. The 17- and 45-kDa proteins, which are also present in greater amounts with the T. pallidum inner membrane protoplasmic cylinder complex, had been previously characterized lipoproteins and are, therefore, not membrane-spanning but rather membrane-anchored by their lipid moiety. In contrast, the 28-, 31-, and 65-kDa proteins are exclusively associated with the outer membrane. Both the purified native and an Escherichia coli recombinant outer membrane form of the 31-kDa protein, designated Tromp1, exhibit porin activity, thereby confirming the membrane-spanning outer membrane topology of Tromp1. The 28-kDa protein, designated Tromp2, has sequence characteristics in common with membrane-spanning outer membrane proteins and has also been recombinantly expressed in E. coli, where it targets exclusively to the E. coli outer membrane. The 65-kDa protein, designated Tromp3, is present in the least amount relative to Tromps1 and 2. Tromps 1, 2, and 3 were antigenic when tested with serum from infection and immune syphilitic rabbits and humans. These newly identified TROMPs provide a molecular foundation for the future study of syphilis pathogenesis and immunity.

Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins.

Purified endoflagella from nonpathogenic Treponema phagedenis biotype Reiter were characterized biochemically and compared antigenically with the endoflagellar proteins of Treponema pallidum. T. phagedenis biotype Reiter endoflagella were dissociated into constituent polypeptides by incubation under conditions which disrupt noncovalent bonds. Chymotrypsin peptide maps of T. phagedenis biotype Reiter endoflagellar proteins revealed that the 37- and 33-kilodalton (kDa) major components shared significant homology with the 27- and 30-kDa minor components, respectively. The peptide maps also suggested that the major components shared a lesser degree of structural similarity with each other. These relationships were confirmed by Western blots of T. phagedenis biotype Reiter endoflagellar proteins employing antibodies that were purified against the individual endoflagellar polypeptides. Western blots of T. pallidum with the purified antibodies also demonstrated strong cross-reactivity between the T. phagedenis biotype Reiter endoflagellar proteins and T. pallidum proteins of identical or similar molecular weights. A unique Western blotting technique that we called epitope bridging was used to determine that the 37-kDa subunit contains most of the external epitopes on T. phagedenis biotype Reiter endoflagella. Immunoelectron microscopy with human syphilitic serum and rabbit T. phagedenis biotype Reiter endoflagellar antiserum confirmed the presence of cross-reactive epitopes on the surface of intact T. phagedenis and T. pallidum endoflagella.

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain.

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.

The antigenic interrelationship between the endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum Nichols strain. I. Treponemicidal activity of cross-reactive endoflagellar antibodies against T. pallidum.

Treponemicidal activity against Treponema pallidum, Nichols strain, by anti-endoflagellar antibodies and the presence of antigenic interrelationships between the endoflagella of Treponema phagedenis biotype Reiter (TPR) and T. pallidum have been demonstrated. SDS-PAGE profiles of purified endoflagella from both organisms were similar, identifying five polypeptide bands for TPR (37,000, 33,000 doublet, 30,000, and 27,000 daltons) and five polypeptide bands for T. pallidum (35,000, 33,000 doublet, 30,000, and 27,000 daltons). Antiserum against TPR endoflagella identified identical bands on Western blots of TPR, T. pallidum, and the respective endoflagellar preparations. Western blots confirmed the presence of antibodies in normal human serum (NHS) against the 33,000 dalton treponemal endoflagellar proteins. The complement-dependent treponemicidal activity of NHS against T. pallidum was completely removed by absorption with purified TPR endoflagella. Furthermore, rabbit antisera against TPR endoflagella were reactive in the Treponema pallidum immobilization (TPI) test. These findings demonstrate that anti-endoflagellar antibodies are treponemicidal against T. pallidum. A possible mechanism for this activity is discussed in relation to the subsurface location of endoflagella.

Antigenic and structural characterization of Treponema pallidum (Nichols strain) endoflagella.

Purified endoflagella from Treponema pallidum, Nichols strain, were characterized both structurally and antigenically. Structural analysis showed T. pallidum endoflagella are composed of 35- and 33-kilodalton (kDa) subunits which lack cysteine and do not share N-terminal amino acid sequence homology (20 residues). Intact endoflagella were dissociated into the composite subunits by incubation, which disrupts noncovalent bonds. Antiserum raised against purified T. pallidum endoflagella identified shared epitopes on the endoflagellar polypeptides of the nonpathogen, Treponema phagedenis biotype Reiter. Pathogen-specific epitopes were also found on the 35- and 33-kDa polypeptides by using affinity-purified endoflagellar antibodies. The pathogen-specific epitopes were localized by immunoblotting analysis of chymotryptic digests of the endoflagellar subunits; 18- and 26-kDa fragments derived from the 35-kDa subunit were found to possess a majority of the pathogen-specific epitopes. Both the 35- and 33-kDa subunits had surface exposure, as determined by immunoelectron microscopy, although additional immunochemical data indicated that the surface exposure of the 35-kDa subunit was greater.

Attachment of Treponema pallidum to fibronectin, laminin, collagen IV, and collagen I, and blockage of attachment by immune rabbit IgG.

As shown by scanning electron and phase contrast microscopy, Treponema pallidum attached in vitro to basement membranes purified from kidney cortex tissues or from retinal vessels. This organism also attached to the extracellular matrix remaining after cultured cells had been solubilised with Triton X. Fibronectin, laminin, collagen, IV, collagen I, and hyaluronic acid are structural components of basement membranes and extracellular matrices. Experiments were performed to investigate the in vitro attachment of T pallidum to each of these components. Viable or heat inactivated treponemes were added to glass coverslips precoated with different concentrations of each component. After various times of incubation, coverslips were washed and the attached organisms were counted. Large numbers of viable organisms attached to each of these five components. In contrast, heat inactivation sharply reduced numbers of attached organisms. The IgG fractions of immune and non-immune rabbit serum samples were affinity purified using protein A. T pallidum was preincubated with both fractions, then incubated with either intact cultured cells or with coverslips coated with the five tissue components. The IgG from immune serum blocked treponemal attachment to the cultured cells and to fibronectin, laminin, collagen IV, and collagen I, but not to hyaluronic acid. These results are discussed in terms of attachment mechanisms of T pallidum and potential applications to in vivo infection.

Protection elicited by native outer membrane protein Oms66 (p66) against host-adapted Borrelia burgdorferi: conformational nature of bactericidal epitopes.

Oms66 is a Borrelia burgdorferi outer membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection with B. burgdorferi (IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using "host-adapted" B. burgdorferi B31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.