RESUMO
UNLABELLED: Since its identification as the causative agent of plague in 1894, thousands of Yersinia pestis strains have been isolated and stored. Here, we report the ability of Y. pestis to survive up to 47 years in agar stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. Specifically, we found variation in the presence of plasmid and chromosomal virulence markers (genes pla, lcrV, caf1 and irp2) among multiple subcultures of Y. pestis strains in the 'Collection of Yersinia pestis' (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil. This variation, together with all of the inherent temporal, geographic and other genetic variation represented by all of the recoverable strains in this historical collection was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the ability of Yersinia pestis to survive up to 47 years in agar stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. This variation, together with all of the inherent temporal, geographic and other genetic variation represented by all of the recoverable strains in the historical 'Collection of Yersinia pestis' (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis.
Assuntos
Ágar/farmacologia , Plasmídeos/genética , Yersinia pestis , Brasil , Criopreservação , Variação Genética , Humanos , Peste/microbiologia , Virulência/genética , Yersinia pestis/genética , Yersinia pestis/crescimento & desenvolvimento , Yersinia pestis/patogenicidadeRESUMO
We subtyped Brazilian Yersinia pestis strains by pulsed-field gel electrophoresis (PFGE). This was done with 22 Brazilian Y. pestis strains: 17 from an outbreak and 5 from endemic routine surveillance. The strains were divided into 2 groups (I and II), 8 subgroups (A-H) and 19 PFGE profiles or pulsotypes. PFGE did not separate outbreak from non-outbreak strains, as identical pulsotype patterns were found among outbreak strains and strains obtained from surveillance. However, it was able to detect intraspecific genetic diversity among Brazilian strains. This PFGE technique was able to differentiate a homogeneous group of Brazilian Y. pestis strains.
Assuntos
Yersinia pestis/classificação , Técnicas de Tipagem Bacteriana , Brasil , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Geografia , Reprodutibilidade dos Testes , Yersinia pestis/genéticaRESUMO
Plague outbreaks are occasionally reported in Brazil. Unfortunately, due to great genetic similarity, molecular subtyping of Yersinia pestis strains is difficult. Analysis of multiple-locus variable number of tandem repeats (VNTR), also known as MLVA, has been found to be a valuable tool to discriminate among strains. To check for genetic differences, strains obtained from two different ecological complexes in Brazil collected during two different epidemiological events, an epizootic in Sítio Alagoinha in 1967 and an outbreak in Planalto da Borborema in 1986, were subtyped through MLVA using 12 VNTR loci. Three clusters (A, B and C) were observed. Of the 20 strains from the epizootic, 18 fit into cluster A. Cluster A was divided into two subgroups: A(1) (15 strains) and A(2) (3 strains). Of the 17 strains from the outbreak, 15 fit into cluster B. Cluster B was divided into three subgroups: B(1) (4 strains), B(2) (4 strains) and B(3) (7 strains). Cluster C is a singleton with one epizootic strain. The external standards, Y. pestis CO92 and Y. pseudotuberculosis IP32953, formed two clusters of singletons. The stability of 12 VNTR loci of three unrelated cultures included in this study was assessed. The 12 VNTR loci were stable through multiple serial subcultures in the laboratory. MLVA revealed that Y. pestis populations in Brazil are not monomorphic, and that there is intraspecific genetic diversity among Brazilian plague strains. We conclude that there is some correlation among genetic groups of this species, related to the temporal and geographic origin of isolates.
Assuntos
Variação Genética , Yersinia pestis/genética , Alelos , Brasil , Análise por Conglomerados , Eletroforese em Gel de Ágar , Loci Gênicos/genética , Geografia , Repetições Minissatélites/genética , Tipagem de Sequências Multilocus , Filogenia , Peste/microbiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Yersinia pestis/classificaçãoRESUMO
Sera from 269 rodents obtained during the routine surveillance operations in plague areas of Rio de Janeiro and Pernambuco states, Brazil were tested by ELISA for specific IgG antibodies against a recombinant nucleocapsid (N) protein of Araraquara hantavirus. ELISA-positive sera were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) for amplification of the virus genome and later sequencing for identification of the viral variant. The samples from the state of Pernambuco were antibody negative, and although four from Rio de Janeiro were ELISA-positive, they failed to yield viral cDNA by RT-PCR. This is the first report of the presence of antibodies to a hantavirus among rodents from Rio de Janeiro and suggests the possibility of human cases of hantavirus pulmonary syndrome (HPS) in that state, although no case has yet been reported.
Assuntos
Animais Selvagens/virologia , Anticorpos Antivirais/sangue , Infecções por Hantavirus/veterinária , Orthohantavírus/imunologia , Doenças dos Roedores/epidemiologia , Sigmodontinae/virologia , Animais , Animais Selvagens/classificação , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática , Orthohantavírus/classificação , Orthohantavírus/genética , Orthohantavírus/isolamento & purificação , Infecções por Hantavirus/epidemiologia , Infecções por Hantavirus/virologia , Imunoglobulina G/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças dos Roedores/virologia , Sigmodontinae/classificaçãoRESUMO
We developed an efficient method to prepare a hybrid inorganic-organic composite based on polyvinyl alcohol (PVA) and polysiloxane using the sol-gel disc technique. Antigen obtained from Yersinia pestis was covalently immobilized onto these discs with glutaraldehyde and used as solid phase in ELISA for antibody detection in serum of rabbits experimentally immunized with plague. Using 1.25 microg antigen per disc, a peroxidase conjugate dilution of 1:4,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. These values are similar to those used for PVA-glutaraldehyde discs, plasticized filter paper discs and the polyaniline-Dacron composite discs. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates, with the amount of antigen being one fourth that employed in conventional PVC plates (5 microg/well). In addition to the performance of the polysiloxane/PVA-glutaraldehyde disc as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Álcool de Polivinil/química , Siloxanas/química , Yersinia/imunologia , Animais , Ensaio de Imunoadsorção Enzimática/instrumentação , Fixadores , Glutaral , CoelhosRESUMO
AIMS: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment. METHODS AND RESULTS: The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971-1997). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.
Assuntos
Peste/microbiologia , Yersinia pestis/classificação , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Brasil , Humanos , Testes de Sensibilidade Microbiana , Pigmentos Biológicos/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase , Roedores/microbiologia , Sifonápteros/microbiologia , Virulência/genética , Yersinia pestis/genética , Yersinia pestis/patogenicidadeRESUMO
AIMS: To determine the effectiveness of multiplex-PCR in Yersinia pestis identification in samples preserved in Cary & Blair medium and to evaluate if this technique would uncover Y. pestis-positives among culture-negative samples. METHODS AND RESULTS: Multiplex-PCR was used to detect Y. pestis in Cary & Blair preserved bubo aspirates from experimentally infected guinea pigs and to re-analyze samples from a plague outbreak after prolonged storage in Cary & Blair. Variation in the target genes amplification was observed over time. CONCLUSIONS: Multiplex-PCR proved to be more effective than culture for plague diagnosis, both for old and recent samples. This technique would be a valuable tool for the plague control programme. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex-PCR technique can be useful for the detection and characterization of Y. pestis even when the bacteria are no longer viable and when culture diagnosis has been hampered by the growth of contaminants.
Assuntos
Peste/diagnóstico , Reação em Cadeia da Polimerase/métodos , Yersinia pestis/isolamento & purificação , Animais , Primers do DNA , DNA Bacteriano/análise , Cobaias , Peste/microbiologia , Sensibilidade e Especificidade , Yersinia pestis/genética , Yersinia pestis/patogenicidadeRESUMO
Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.
Assuntos
DNA Bacteriano/isolamento & purificação , Instabilidade Genômica/genética , Plasmídeos/genética , Yersinia pestis/patogenicidade , Animais , Western Blotting , DNA Bacteriano/genética , Eletroforese em Gel de Ágar , Genoma Bacteriano , Humanos , Dose Letal Mediana , Camundongos , Reação em Cadeia da Polimerase , Virulência/genética , Yersinia pestis/genéticaRESUMO
Most Brazilian Yersinia pestis isolates display a typical plasmid profile composed of the three classical plasmids: pYV, pPst and pFra. However, some cultures lack at least one of these plasmids, while a few of them harbour atypical DNA bands of molecular weight ranging from 147 to 11.5 kb. To investigate whether Y. pestis displaying atypical plasmid content could be propagated among rodents in nature through flea bites, we carried out studies with fleas ( Xenopsylla cheopis) and rodents ( Calomys callosus) reared in the laboratory and five Y. pestis cultures differing in plasmid content. The results suggest that: (1) the single presence of pYV is not sufficient for the transmission of Y. pestis by fleas, (2) pPst is not essential for transmission, (3) two atypical DNA bands of molecular weight of 30 kb and >90 kb have no biological role, and (4) pFra is required for the transmission of Y. pestis by flea bites. Other studies are needed to determine whether this plasmid alone is sufficient for transmission.
Assuntos
Muridae/microbiologia , Peste/transmissão , Plasmídeos/genética , Sifonápteros/microbiologia , Yersinia pestis/genética , Animais , Meios de Cultura , Interações Hospedeiro-Parasita , Camundongos , Plasmídeos/fisiologia , Yersinia pestis/crescimento & desenvolvimentoRESUMO
AIMS: To investigate genetic diversity among Staphylococcus aureus and to delineate the geographical distribution of the strains found. METHODS AND RESULTS: RAPD-PCR and ribotyping-PCR were employed for the characterization of Staph. aureus isolates from bovine and nosocomial origin. Among the strains, five to nine groups were distinguished by RAPD-PCR, depending on which primer was used, while ribotyping-PCR distinguished seven ribotypes. CONCLUSIONS, AND SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the genetic heterogeneity of the strains studied, and the large dissemination of some clones throughout different regions and hosts, findings that may allow the monitoring of Staph. aureus infections.
Assuntos
Técnicas de Tipagem Bacteriana , Técnica de Amplificação ao Acaso de DNA Polimórfico , Ribotipagem , Staphylococcus aureus/genética , Animais , Bovinos , Impressões Digitais de DNA , Ecologia , Heterogeneidade Genética , Humanos , Reação em Cadeia da Polimerase , Estudos Soroepidemiológicos , Staphylococcus aureus/classificaçãoRESUMO
AIMS: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates. MATERIALS AND RESULTS: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found. CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.
Assuntos
Cólera/epidemiologia , DNA Bacteriano/análise , Técnica de Amplificação ao Acaso de DNA Polimórfico , Vibrio cholerae O1/genética , Brasil/epidemiologia , Cólera/microbiologia , Bases de Dados Genéticas , Humanos , Prática de Saúde PúblicaRESUMO
Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.
Assuntos
Humanos , Animais , Camundongos , DNA Bacteriano , Genoma Bacteriano , Instabilidade Genômica , Plasmídeos , Yersinia pestis , Western Blotting , Meios de Cultura , DNA Bacteriano , Eletroforese em Gel de Ágar , Dose Letal Mediana , Reação em Cadeia da Polimerase , Virulência , Yersinia pestisRESUMO
We developed an efficient method to prepare a hybrid inorganic-organic composite based on polyvinyl alcohol (PVA) and polysiloxane using the sol-gel disc technique. Antigen obtained from Yersinia pestis was covalently immobilized onto these discs with glutaraldehyde and used as solid phase in ELISA for antibody detection in serum of rabbits experimentally immunized with plague. Using 1.25 æg antigen per disc, a peroxidase conjugate dilution of 1:4,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. These values are similar to those used for PVA-glutaraldehyde discs, plasticized filter paper discs and the polyaniline-Dacron composite discs. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates, with the amount of antigen being one fourth that employed in conventional PVC plates (5 æg/well). In addition to the performance of the polysiloxane/PVA-glutaraldehyde disc as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application
Assuntos
Animais , Coelhos , Ensaio de Imunoadsorção Enzimática , Antígenos de Bactérias , Ensaio de Imunoadsorção Enzimática , Fixadores , Glutaral , Álcool de Polivinil , YersiniaRESUMO
F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3 percent w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.
Assuntos
Animais , Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Peste/diagnóstico , Álcool de Polivinil/farmacologia , Yersinia pestis/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/sangue , Ensaio de Imunoadsorção Enzimática/economia , Ensaio de Imunoadsorção Enzimática/instrumentação , Cabras , Peste/imunologia , CoelhosRESUMO
Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 µg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Celulose , Peste/diagnóstico , Yersinia pestis/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G , TitulometriaRESUMO
The distribution of Yersina pestis Fraction-1 (F1) antigen was analyzed in cells grown at 28§C and 37§C. Fractionation of Y. pestis cells followed by analysis in SDS-polyacrylamide gel electrophoresis indicated that the mature form of the F1 antigen is localized in the extracellular matrix and in the cytoplasm. Localization of the F1 antigen was confirmed by immunoblots and a single peptide with a molecular weight of 17,000 daltons was recognized. Electron microscopy of Y. pestis cells labeled with colloidal gold-conjugated antibodies corroborated the extracellular matrix and cytoplasm dual location of the F1 antigen