RESUMEN
Spiroplasma spp., tiny filterable wall-less bacteria, are consistently associated with the transmissible spongiform encephalopathies (TSE). Spiral forms have been transiently isolated from TSE-affected brain tissues in SP4 growth media designed for isolation of Spiroplasma spp., but the isolate could not be propagated in SP4 media. A bacterium must grow in vitro in cell-free cultures to allow full characterization of a suspect pathogen. Here, a novel Spiroplasma sp. was isolated from scrapie- and chronic wasting disease (CWD)-affected brains and lymph nodes. Filtrates of tissue homogenates inoculated into Brucella media incubated for 14 days at 35 °C resulted in high titers of spiroplasma as shown by dark-field microscopy. A drop assay of infected media on Bacto Schaedler agar showed spiroplasma isolates forming unique subsurface colonies after 21 days incubation. Spiroplasma coils, coccoid forms and clumps of entwined spiroplasma filaments were seen on the agar by scanning electron microscopy. Since Brucella media has a sodium bisulfite additive that lowers oxygen tension, TSE spiroplasma growth requires media with low oxygen tension. Brucella media allows for isolation and propagation of spiroplasma from TSE-affected tissues, which will lead to complete characterization of this TSE pathogen and determine its role as a candidate causative agent of TSE.
Asunto(s)
Encéfalo/microbiología , Ganglios Linfáticos/microbiología , Enfermedades por Prión/microbiología , Spiroplasma/aislamiento & purificación , Animales , Encéfalo/patología , Enfermedades por Prión/patología , OvinosRESUMEN
OBJECTIVE: Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model. PROCEDURE: The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE). RESULTS: The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie-affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA. CONCLUSION: These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.
Asunto(s)
Infecciones por Bacterias Gramnegativas/veterinaria , Enfermedades de la Retina/veterinaria , Scrapie/complicaciones , Spiroplasma , Animales , Células Cultivadas , Ojo/microbiología , Ojo/patología , Técnica del Anticuerpo Fluorescente Directa/veterinaria , Infecciones por Bacterias Gramnegativas/complicaciones , Infecciones por Bacterias Gramnegativas/microbiología , Microscopía Electrónica/veterinaria , Retina/microbiología , Retina/patología , Enfermedades de la Retina/complicaciones , Enfermedades de la Retina/microbiología , Enfermedades de la Retina/patología , Scrapie/microbiología , OvinosRESUMEN
We report on the presence of specific antibodies to Brucella spp. and Yersinia enterocolitica in polar bears (Ursus maritimus) from northern Alaska (southern Beaufort Sea) during 2003-2006. Based on numerous known stressors (e.g., climate change and loss of sea ice habitat, contaminants), there is increased concern regarding the status of polar bears. Considering these changes, it is important to assess exposure to potentially pathogenic organisms and to improve understanding of transmission pathways. Brucella or specific antibodies to Brucella spp. has been reported in marine mammals. Various assays were used to elucidate the pathway or source of exposure (e.g., "marine" vs. "terrestrial" Brucella spp.) of northern Alaska polar bears to Brucella spp. The standard plate test (SPT) and the buffered Brucella antigen card test (BBA) were used for initial screening for antibodies specific to Brucella. We then evaluated positive reactors (presence of serum antibody specific for Brucella spp.) using immunoblots and competitive enzyme-linked immunosorbent assay (cELISA; based on pinniped-derived Brucella spp. antigen). Annual prevalence of antibody (BBA and SPT) for Brucella spp. ranged from 6.8% to 18.5% over 2003-2006, with an overall prevalence of 10.2%. Prevalence of Brucella spp. antibody did vary by age class. Western blot analyses indicated 17 samples were positive for Brucella spp. antibody; of these, 13 were negative by marine (pinniped) derived Brucella antigen cELISA and four were positive by marine cELISA. Of the four samples positive for Brucella antibody by marine cELISA, three cross-reacted with Y. enterocolitica and Brucella spp. (one sample was Brucella negative and Y. enterocolitica positive). It appears the polar bear antibody does not react with the antigens used on the marine cELISA assay, potentially indicating a terrestrial (nonpinniped) source of Brucella spp.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Brucella/inmunología , Ursidae/microbiología , Yersinia enterocolitica/inmunología , Alaska/epidemiología , Animales , Femenino , Masculino , Estudios Seroepidemiológicos , Estrés PsicológicoRESUMEN
With the completion of the genomic sequence of Brucella melitensis 16M, a putative hemagglutinin gene was identified which is present in 16M and absent in Brucella abortus. The possibility of this hemagglutinin being a potential virulence factor was evaluated via gene replacement in B. melitensis yielding 16MΔE and expression in trans in B. abortus 2308-QAE. Utilizing the caprine brucellosis model, colonization and pathogenesis studies were performed to evaluate these strains. B. melitensis 16M hemagglutinin gene expression in trans in 2308-QAE revealed a significant (p≤0.05) increase in colonization and abortion rates when compared to B. abortus 2308, mimicking B. melitensis 16M virulence in pregnant goats. The B. melitensis disruption mutant's colonization and abortion rates demonstrated no attenuation in colonization but displayed a 28% reduction in abortions when compared to parental B. melitensis 16M.
Asunto(s)
Proteínas Bacterianas/genética , Brucella melitensis/genética , Brucelosis/veterinaria , Enfermedades de las Cabras/microbiología , Hemaglutininas/genética , Aborto Veterinario/microbiología , Animales , Anticuerpos Antibacterianos/sangre , Brucella abortus/genética , Brucella melitensis/patogenicidad , Brucelosis/microbiología , ADN Bacteriano/genética , Femenino , Cabras , Mutación , Embarazo , VirulenciaRESUMEN
Spiroplasma, small motile wall-less bacteria, are linked by molecular and serological studies to the transmissible spongiform encephalopathies (TSEs), which include scrapie in sheep, chronic wasting disease (CWD) in deer and Creutzfeldt-Jakob disease in humans. In this study, two experiments were undertaken to determine the role of spiroplasma in the pathogenesis of TSE. In experiment 1, Spiroplasma mirum, a rabbit tick isolate that had previously been shown to experimentally induce spongiform encephalopathy in rodents, was inoculated intracranially (IC) into ruminants. S. mirum-inoculated deer manifested clinical signs of TSE after 1.5 to 5.5 months incubation. The deer, as well as sheep and goats, inoculated with S. mirum developed spongiform encephalopathy in a dose-dependent manner. In experiment 2, spiroplasma closely related to S. mirum were isolated from TSE-affected brains via passage in embryonated eggs, and propagated in cell-free M1D media. Spiroplasma spp. isolates from scrapie-affected sheep brain and from CWD-affected deer brain inoculated IC into sheep and goats induced spongiform encephalopathy closely resembling natural TSE in these animals. These data show spiroplasma to be consistently associated with TSE, and able experimentally to cause TSE in ruminant animal models, therein questioning the validity of studies that have concluded the prion, a miss-folded protease-resistant protein that builds up in TSE brains during the course of the disease, to be the sole causal agent. The spiroplasma infection models reported here will be important for investigating factors involved in the pathogenesis of TSE since ruminants are the natural hosts.
Asunto(s)
Encéfalo/microbiología , Enfermedades por Prión/veterinaria , Rumiantes/microbiología , Spiroplasma/aislamiento & purificación , Spiroplasma/patogenicidad , Garrapatas/microbiología , Animales , Ciervos , Enfermedades de las Cabras/microbiología , Cabras , Sistemas Multiinstitucionales , Enfermedades por Prión/microbiología , Enfermedades por Prión/transmisión , Ovinos , Enfermedades de las Ovejas/microbiologíaRESUMEN
Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.
Asunto(s)
Brucella melitensis/genética , Brucelosis/microbiología , Genes Bacterianos , Enfermedades de las Cabras/microbiología , Animales , Proteínas Bacterianas/genética , Brucella melitensis/crecimiento & desarrollo , Brucella melitensis/patogenicidad , ADN Helicasas/genética , Cabras , Hígado/microbiología , Ganglios Linfáticos/microbiología , Mutagénesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Bazo/microbiología , VirulenciaRESUMEN
Pregnant goats were employed to assess unmarked deletion mutant vaccine candidates BMDeltaasp24, BMDeltacydBA, and BMDeltavirB2, as the target host species naturally infected with Brucella melitensis. Goats were assessed for the degree of pathology associated with the vaccine strains as well as the protective immunity afforded by each strain against abortion and infection after challenge with wild-type Brucella melitensis 16M. Both BMDeltaasp24 and BMDeltavirB2 were considered safe vaccine candidates in the pregnant goat model because they did not cause abortion or colonize fetal tissues. BMDeltaasp24 was isolated from the maternal tissues only, indicating a slower rate of clearance of the vaccine strain than for BMDeltavirB2, which was not isolated from any maternal or fetal tissues. Both strains were protective against abortion and against infection in the majority of pregnant goats, although BMDeltaasp24 was more efficacious than BMDeltavirB2 against challenge infection.
Asunto(s)
Vacuna contra la Brucelosis/inmunología , Brucella melitensis/inmunología , Brucelosis/prevención & control , Vacunas Sintéticas/inmunología , Animales , Vacuna contra la Brucelosis/efectos adversos , Modelos Animales de Enfermedad , Femenino , Cabras , Mutación , Embarazo , VacunaciónRESUMEN
An isogenic katE mutant derived from virulent Brucella melitensis 16M displays hypersensitivity to hydrogen peroxide in disk sensitivity assays but retains the capacity to colonize pregnant goats and induce abortion. These experimental findings indicate that although the sole periplasmic catalase of Brucella melitensis functions as an antioxidant, this enzyme does not play a critical role in virulence in the natural host.
Asunto(s)
Aborto Veterinario/microbiología , Brucella melitensis/enzimología , Brucella melitensis/patogenicidad , Brucelosis/veterinaria , Catalasa/fisiología , Enfermedades de las Cabras/microbiología , Feto Abortado/microbiología , Aborto Veterinario/patología , Animales , Animales Recién Nacidos/microbiología , Brucella melitensis/genética , Brucelosis/microbiología , Brucelosis/patología , Catalasa/genética , Catalasa/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Femenino , Cabras , Peróxido de Hidrógeno/metabolismo , Mutagénesis Insercional , Embarazo , VirulenciaRESUMEN
Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.
Asunto(s)
Brucella abortus/genética , Cromosomas Bacterianos/genética , Recombinación Genética , Animales , Inversión Cromosómica , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.
Asunto(s)
Brucella abortus/patogenicidad , Regulación Bacteriana de la Expresión Génica , Hidroxibenzoatos/metabolismo , Hierro/metabolismo , Operón , Complicaciones Infecciosas del Embarazo/veterinaria , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Brucella abortus/genética , Brucelosis Bovina/microbiología , Brucelosis Bovina/fisiopatología , Bovinos , Escherichia coli/genética , Femenino , Prueba de Complementación Genética , Datos de Secuencia Molecular , Familia de Multigenes , Embarazo , Complicaciones Infecciosas del Embarazo/microbiología , VirulenciaRESUMEN
Brucella melitensis is a facultative, intracellular, gram-negative cocco-bacillus that causes Malta fever in humans and brucellosis in animals. There are at least six species in the genus, and the disease is classified as zoonotic because several species infect humans. Using 2-D gel electrophoresis and mass spectrometry, we have initiated (i) a comprehensive mapping and identification of all the expressed proteins of B. melitensis virulent strain 16M, and (ii) a comparative study of its proteome with the attentuated vaccinal strain Rev 1. Comprehensive proteome maps of all six Brucella species will be generated in order to obtain vital information for vaccine development, identification of pathogenicity islands, and establishment of host specificity and evolutionary relatedness.
Asunto(s)
Proteínas Bacterianas/metabolismo , Vacuna contra la Brucelosis , Brucella melitensis , Proteoma/análisis , Animales , Proteínas Bacterianas/genética , Vacunas Bacterianas , Brucella/genética , Brucella/metabolismo , Brucella/patogenicidad , Brucella melitensis/genética , Brucella melitensis/metabolismo , Brucella melitensis/patogenicidad , Brucelosis/prevención & control , Brucelosis/veterinaria , Mapeo Cromosómico/veterinaria , Electroforesis en Gel Bidimensional/veterinaria , Regulación Bacteriana de la Expresión Génica , Humanos , Especificidad de la Especie , Virulencia , ZoonosisRESUMEN
Filarial infections have been associated with the development of a strongly polarized Th2 host immune response and a severe impairment of mitogen-driven proliferation and type 1 cytokine production in mice and humans. The role of this polarization in the development of the broad spectra of clinical manifestations of lymphatic filariasis is still unknown. Recently, data gathered from humans as well as from immunocompromised mouse models suggest that filariasis elicits a complex host immune response involving both Th1 and Th2 components. However, responses of a similar nature have not been reported in immunologically intact permissive models of Brugia infection. Brucella abortus-killed S19 was inoculated into the Brugia-permissive gerbil host to induce gamma interferon (IFN-gamma) production. Gerbils were then infected with B. pahangi, and the effect of the polarized Th1 responses on worm establishment and host cellular response was measured. Animals infected with both B. abortus and B. pahangi showed increased IFN-gamma and interleukin-10 (IL-10) and decreased IL-4 and IL-5 mRNA levels compared with those in animals infected with B. pahangi alone. These data suggest that the prior sensitization with B. abortus may induce a down regulation of the Th2 response associated with Brugia infection. This reduced Th2 response was associated with a reduced eosinophilia and an increased neutrophilia in the peritoneal exudate cells. The changes in cytokine and cellular environment did not inhibit the establishment of B. pahangi intraperitoneally. The data presented here suggest a complex relationship between the host immune response and parasite establishment and survival that cannot be simply ascribed to the Th1/Th2 paradigm.
Asunto(s)
Brucella abortus/inmunología , Brugia pahangi/inmunología , Citocinas/genética , Filariasis/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Eosinófilos/fisiología , Gerbillinae , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-10/genética , Interleucina-4/genética , Interleucina-5/genética , Masculino , ARN Mensajero/análisis , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.
Asunto(s)
Proteínas Bacterianas/análisis , Brucella melitensis/química , Proteoma/análisis , Animales , Cromosomas Bacterianos , Electroforesis en Gel Bidimensional , Genes Bacterianos , Humanos , Focalización Isoeléctrica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.
Asunto(s)
Proteínas Bacterianas/metabolismo , Vacuna contra la Brucelosis , Brucella melitensis/metabolismo , Proteoma/análisis , Proteínas Bacterianas/genética , Brucella melitensis/genética , Brucella melitensis/crecimiento & desarrollo , Brucella melitensis/patogenicidad , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , VirulenciaRESUMEN
Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.