RESUMO
A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.
Assuntos
Aeromonas hydrophila/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/isolamento & purificação , Aeromonas hydrophila/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Infecções por Bactérias Gram-Negativas/microbiologia , Células HT29 , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMO
Host immune mechanisms were proposed to decline under microgravity conditions during spaceflights, which might result in severe infections in astronauts. Therefore, it was important to investigate the effects of microgravity on infecting organisms and their interaction with host cells. Data showed that simulated microgravity (SMG) conditions markedly increased production of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin, which induced fluid secretory responses in a mouse model. SMG also enhanced production of tumor necrosis factor-alpha in murine macrophages infected with enteropathogenic E. coli (EPEC). In a similar fashion, simulated microgravity conditions augmented the invasive potential of Salmonella enterica serovar typhimurium and enhanced production of tumor necrosis-factor alpha in S. typhimurium-infected epithelial cells. Furthermore, coculturing of macrophages and S. typhimurium in a simulated microgravity environment resulted in activation of stress-associated mitogen-activated protein kinase kinase 4. Using the antiorthostatic tail suspension mouse model, which simulates some aspects of microgravity, oral inoculation of S. typhimurium markedly reduced the 50% lethal dose compared to mice infected under normal gravitational conditions. Microarray analysis revealed simulated microgravity-induced alterations in the expression of 22 genes in S. typhimurium, and protein expression profiles were altered in both EPEC and S. typhimurium, based on two-dimensional gel electrophoresis. These studies indicated alterations in the virulence potential of bacteria and in host responses to these pathogens under simulated microgravity conditions, which may represent an important environmental signal. Such studies are essential for better understanding bacterial-host cell interactions, particularly in the context of spaceflights and space habitations of long duration.
Assuntos
Escherichia coli/patogenicidade , Salmonelose Animal/fisiopatologia , Salmonella typhimurium/patogenicidade , Simulação de Ausência de Peso/efeitos adversos , Animais , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Dinoprostona/análise , Enterobacteriaceae/patogenicidade , Proteínas de Escherichia coli/biossíntese , Genes Bacterianos/fisiologia , Humanos , MAP Quinase Quinase 4/imunologia , Macrófagos/microbiologia , Camundongos , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Fator de Necrose Tumoral alfa/análise , Virulência/genética , Virulência/fisiologiaRESUMO
Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.
Assuntos
Expressão Gênica/fisiologia , Lipoproteínas/genética , Lipoproteínas/metabolismo , Análise em Microsséries , Salmonella typhimurium/metabolismo , Flagelos/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mutação , Salmonella typhimurium/classificação , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/ultraestrutura , Sorologia , Virulência/genética , Virulência/fisiologiaRESUMO
We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.
Assuntos
Proteínas de Bactérias/genética , Lipoproteínas/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Aciltransferases/genética , Animais , Proteínas de Bactérias/imunologia , Deleção de Genes , Soros Imunes/imunologia , Lipopolissacarídeos/imunologia , Lipoproteína(a)/genética , Lipoproteína(a)/imunologia , Lipoproteínas/imunologia , Camundongos , Mutação , Infecções por Salmonella/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologiaRESUMO
We performed microarray analyses on RNA from human intestinal epithelial (HT-29) cells treated with the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to examine global cellular transcriptional responses. Based on three independent experiments, Act upregulated the expression of 34 genes involved in cell growth, adhesion, signaling, immune responses (including interleukin-8 [IL-8] production), and apoptosis. We verified the upregulation of 14 genes by real-time reverse transcriptase-PCR and confirmed Act-induced production of IL-8 by enzyme-linked immunosorbent assay on supernatants from nonpolarized and polarized HT-29 cells. Maximal production of IL-8 in response to Act required the presence of intracellular calcium, since chelation of calcium with BAPTA-AM significantly reduced Act-induced IL-8 production in HT-29 cells. We also examined activation of mitogen-activated protein kinases and, as demonstrated by Western blot analysis of apical side-treated polarized HT-29 cells, Act induced phosphorylation of p38, c-Jun NH(2)-terminal kinase, and extracellular signal-regulated kinase 1/2. In addition, KinetWorks proteomics screening of whole-cell lysates revealed Act-induced phosphorylation of cyclic AMP-response element binding protein (CREB), c-Jun, adducin, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) and decreased phosphorylation of protein kinase Balpha, v-raf-1 murine leukemia viral oncogene homolog 1 (i.e., Raf1), and STAT1. We verified activation of CREB and activator protein 1 in polarized cells by gel shift assay. This is the first description of human intestinal epithelial cell transcriptional alterations, phosphorylation or activation of signaling molecules, cytokine production, and calcium mobilization in response to this toxin.
Assuntos
Proteínas de Bactérias/farmacologia , Enterotoxinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/metabolismo , Proteômica , Aeromonas hydrophila/metabolismo , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/metabolismo , Linhagem Celular , Enterotoxinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Intestinos/citologia , Proteínas/genéticaRESUMO
Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-alpha and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Citocinas/metabolismo , Humanos , Lipoproteínas/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Virulência/genética , Virulência/fisiologiaRESUMO
We performed microarray analyses of murine peritoneal macrophages to examine cellular transcriptional responses to a cytotoxic enterotoxin of Aeromonas hydrophila. While 66% of altered genes were common to both primary macrophages and the murine macrophage cell line RAW 264.7, Act caused expression changes of 28 genes specifically in murine peritoneal macrophages.
Assuntos
Aeromonas hydrophila/metabolismo , Proteínas de Bactérias/farmacologia , Enterotoxinas/farmacologia , Regulação da Expressão Gênica , Macrófagos Peritoneais/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Proteínas/genéticaRESUMO
Septic shock due to Salmonella and other gram-negative enteric pathogens is a leading cause of death worldwide. The role of lipopolysaccharide in sepsis is well studied; however, the contribution of other bacterial outer membrane components, such as Braun (murein) lipoprotein (Lpp), is not well defined. The genome of Salmonella enterica serovar Typhimurium harbors two copies of the lipoprotein (lpp) gene. We constructed a serovar Typhimurium strain with deletions in both copies of the lpp gene (lpp1 and lpp2) by marker exchange mutagenesis. The integrity of the cell membrane and the secretion of the effector proteins through the type III secretion system were not affected in the lpp double-knockout mutant. Subsequently, the virulence potential of this mutant was examined in a cell culture system using T84 intestinal epithelial and RAW264.7 macrophage cell lines and a mouse model of salmonellosis. The lpp double-knockout mutant was defective in invading and inducing cytotoxic effects in T84 and RAW264.7 cells, although binding of the mutant to the host cell was not affected when compared to the wild-type (WT) serovar Typhimurium. The motility of the mutant was impaired, despite the finding that the number of flagella was similar in the lpp double knockout mutant and the WT serovar Typhimurium. Deletion in the lpp genes did not affect the intracellular survival and replication of Salmonella in macrophages and T84 cells. Induction of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-8 (IL-8) was significantly reduced in macrophages and T84 cells infected with the lpp double-knockout mutant. The levels of IL-8 remained unaffected in T84 cells when infected with either live or heat-killed WT and lpp mutant, indicating that invasion was not required for IL-8 production and that Toll-like receptor 2 signaling might be affected in the Lpp double-knockout mutant. These effects of the Lpp protein could be restored by complementation of the isogenic mutant. The lpp double-knockout mutant was avirulent in mice, and animals infected with this mutant were protected from a lethal challenge dose of WT serovar Typhimurium. The severe combined immunodeficient mice, on the other hand, were susceptible to infection by the lpp double-knockout mutant. The serovar Typhimurium mutants from which only one of the lpp (lpp1 or lpp2) genes was deleted were also avirulent in mice. Taken together, our data indicated that Lpp specifically contributed to the virulence of the organism.
Assuntos
Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Salmonella typhimurium/patogenicidade , Virulência/fisiologia , Animais , Clonagem Molecular , Citocinas/metabolismo , Deleção de Genes , Lipoproteínas/genética , Camundongos , Camundongos SCID , Peptidoglicano/genética , Salmonella typhimurium/genética , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.
Assuntos
Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/genética , Enterotoxinas/genética , Regulação Bacteriana da Expressão Gênica , Aeromonas hydrophila/genética , Animais , Proteínas de Bactérias/fisiologia , Sequência de Bases , Clonagem Molecular , Elementos de DNA Transponíveis , Feminino , Teste de Complementação Genética , Camundongos , Dados de Sequência Molecular , Mutação , Transcrição Gênica , VirulênciaRESUMO
AIMS: To evaluate the ability of Salmonella enterica ser. Enteritidis outer membrane proteins (OMPs) of 75.6 and 82.3 kDa to inhibit or reduce in vivo colonization of S. Enteritidis on intestinal mucosa in chickens. METHODS AND RESULTS: Nine-week-old specific-pathogen-free chickens were subcutaneously immunized with 75.6 or 82.3 kDa protein, and challenged with a virulent strain of S. Enteritidis. Chickens were killed, and portions of small intestine and caecum were removed at necropsy. The population of S. Enteritidis attached to chicken intestinal mucosa was determined. The population of S. Enteritidis recovered from the small intestine and caecum of chickens immunized with 75.6 or 82.3 kDa protein was significantly (P < 0.05) lower than that recovered from the control birds. CONCLUSIONS: Salmonella Enteritidis OMPs 75.6 kDa and 82.3 kDa were effective in reducing colonization of S. Enteritidis on intestinal mucosa in chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella Enteritidis OMPs 75.6 or 82.3 kDa could be used as potential vaccines to reduce S. Enteritidis colonization in chickens.
Assuntos
Proteínas da Membrana Bacteriana Externa/administração & dosagem , Galinhas/microbiologia , Salmonella enteritidis/crescimento & desenvolvimento , Vacinação/métodos , Animais , Western Blotting/métodos , Ceco/microbiologia , Mucosa Intestinal/microbiologia , Intestino Delgado/microbiologiaRESUMO
AIMS: To express and identify the Salmonella enterica ser. Enteritidis outer membrane proteins that are involved in the attachment to human intestinal epithelial cells. METHODS AND RESULTS: Salmonella Enteritidis attachment proteins were expressed by infection of human intestinal epithelial cells (int-407) with Salm. Enteritidis strain SE28, followed by pulse labelling with [35S]-methionine. Outer membrane proteins were separated by TritonX-114 phase partitioning, and were detected by Salm. Enteritidis-specific polyclonal antibody. Outer membrane proteins of molecular weights 82.3, 75.6, 49.3, 35.5 and 19.3 kDa were newly synthesized. Expression of 31.2 and 16 kDa proteins was up-regulated, whereas the expression of 40.7 kDa protein was down-regulated. Polyclonal antibodies against the 82.3 and 75.6 kDa proteins significantly (P < 0.05) reduced the binding of Salm. Enteritidis to int-407 cells in vitro. CONCLUSIONS: Outer membrane proteins 82.3 and 75.6 kDa are potentially involved in the attachment of Salm. Enteritidis to the intestinal mucosa. SIGNIFICANCE AND IMPACT OF THE STUDY: Outer membrane proteins 82.3 and 75.6 kDa identified in this study could be used as potential vaccines to block or reduce Salm. Enteritidis colonization in chickens.
Assuntos
Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/citologia , Salmonella enteritidis/patogenicidade , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Linhagem Celular , Humanos , Infecções por Salmonella/microbiologia , Salmonella enteritidis/metabolismo , Salmonella enteritidis/fisiologiaRESUMO
This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mphi). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mphi and replicate within the cytoplasm of these cells. Release from the Mphi occurs 4-6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mphi, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mphi release of anthrax bacilli is atxA regulated. The putative 'escape' genes may be located on the chromosome and/or on pXO1.
Assuntos
Antraz/microbiologia , Bacillus anthracis/patogenicidade , Macrófagos/microbiologia , Animais , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/fisiologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Permeabilidade da Membrana Celular , Cromo/metabolismo , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Fagossomos/microbiologia , Plasmídeos/genética , Esporos Bacterianos/fisiologia , Transativadores/metabolismo , VirulênciaRESUMO
A Proteus mirabilis-specific polymerase chain reaction (PCR) was developed and standardized. The origin of the primers was a recombinant clone that contained P. mirabilis-specific Hind III fragment DNA of 3.5-kilobase pairs. Based on the sequence data of P. mirabilis recombinant clone, two primers designated MMKAP 1 and MMKAP 2 were synthesized for use in the PCR. A P. mirabilis-specific 3.5-kb pair DNA product was amplified by the primers from 18 strains of P. mirabilis, but not from other Protease species and bacteria. The minimum amount of target DNA detected by P. mirabilis PCR was 10 fg using ethidium bromide/ultraviolet exposure of gels or Southern blot hybridization with a P. mirabilis recombinant DNA probe.
Assuntos
Primers do DNA , DNA Bacteriano/genética , Reação em Cadeia da Polimerase , Proteus mirabilis/genética , Bactérias/genética , Técnicas de Tipagem Bacteriana , Southern Blotting , Eletroforese em Gel de Ágar , Proteus/classificação , Proteus/genética , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.
Assuntos
Aviadenovirus/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , Animais , Embrião de Galinha , DNA Viral/análise , Eletroforese em Gel de Ágar/veterinária , Sensibilidade e EspecificidadeRESUMO
A reverse transcriptase-polymerase chain reaction method was developed for the detection of avian reovirus. The origin of primers was from the S1 gene of the avian reovirus genome. A reovirus-specific 532-base pair cDNA product was amplified by these primers from six reference strains and 23 field isolates of avian reoviruses, but not from seven different avian pathogenic viruses and bacteria. As little as 1 pg of avian reovirus RNA was detected using gel electrophoresis and Southern blot hybridization.
Assuntos
Orthoreovirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Doenças das Aves Domésticas , Infecções por Reoviridae/veterinária , Animais , Embrião de Galinha , Galinhas , Primers do DNA , Fibroblastos , Orthoreovirus/classificação , Orthoreovirus/genética , DNA Polimerase Dirigida por RNA , Infecções por Reoviridae/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Salmonella enteritidis isolates of known phage types 8, 13a, and 14b were inoculated separately into a group of 14-wk-old specific-pathogen-free chickens. S. enteritidis isolates from pre- and postinoculum were analyzed with the use of arbitrarily primed polymerase chain reaction to determine any genotypic changes after reisolation from the chickens. The preinoculum isolates and the isolates of S. enteritidis recovered at 5, 10, 20, and 35 days postinoculation from the inoculated chickens with various phage types were similar. Therefore, no changes of S. enteritidis organisms of similar phage types occurred at the genotypic level. However, SE-3-B7001 (phage type 8) was changed to phage type 14b after inoculation into the chickens.
Assuntos
Fagos de Salmonella/isolamento & purificação , Salmonella enteritidis/genética , Animais , Tipagem de Bacteriófagos , Galinhas , DNA Bacteriano/isolamento & purificação , Genótipo , Reação em Cadeia da Polimerase/métodos , Salmonelose Animal/microbiologia , Fagos de Salmonella/classificação , Salmonella enteritidis/classificação , Salmonella enteritidis/virologia , Organismos Livres de Patógenos EspecíficosRESUMO
Mycoplasma infections are of great concern in avian medicine, because they cause economic losses in commercial poultry production. A multiplex polymerase chain reaction (PCR) was optimized to simultaneously detect four pathogenic species of avian mycoplasmas. Four sets of oligonucleotide primers specific for Mycoplasma gallisepticum (MG), M. synoviae (MS), M. meleagridis (MM) and M. iowae (MI) were used in the test. By using agarose gel electrophoreses for detection of the PCR-amplified DNA products, the sensitivity of detection was between 1 pg for MG, 1 pg for MS, 100 fg for MM and 100 pg for MI after 35 cycles of PCR. Similar sensitivity of these primers was achieved with broth cultures of these four organisms.
Assuntos
DNA Bacteriano/isolamento & purificação , Mycoplasma/genética , Animais , Doenças das Aves/microbiologia , Primers do DNA , Eletroforese em Gel de Ágar , Marcadores Genéticos , Mycoplasma/isolamento & purificação , Infecções por Mycoplasma/veterinária , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
An arbitrarily primed PCR (AP-PCR) was developed to analyze the genomic DNAs of Salmonella enteritidis isolates from human outbreaks and from avian sources. The AP-PCR generated seven distinct randomly amplified DNA patterns among the S. enteritidis isolates studied. Differences in the DNA patterns among isolates of S. enteritidis phage types 13a and 8 as well as among S. enteritidis phage type 14b were observed. The AP-PCR analysis can be used to determine the differences among isolates within the same phage types and may be useful for tracing back the source of S. enteritidis outbreaks in humans more precisely.
Assuntos
Reação em Cadeia da Polimerase/métodos , Salmonella enteritidis/genética , Salmonella enteritidis/isolamento & purificação , Animais , Técnicas Bacteriológicas , Sequência de Bases , Doenças das Aves/microbiologia , Aves/microbiologia , Primers do DNA/genética , DNA Bacteriano/genética , Surtos de Doenças , Estudos de Avaliação como Assunto , Humanos , Dados de Sequência Molecular , Infecções por Salmonella/epidemiologia , Infecções por Salmonella/microbiologia , Salmonelose Animal/microbiologia , Fagos de Salmonella/classificação , Fagos de Salmonella/genética , Salmonella enteritidis/virologiaRESUMO
53.7% of the already parasitologically proved schistosomiasis cases showed circulating schistosomal antigen (C.S.A.) in their sera with significantly higher levels than the controls, with no correlation between the level of C.S.A. and the foecal egg count. Significant higher levels of schistosomal complement C3 were found in schistosomiasis patients as compared to the control group, with no significant difference in the mean level of C3 between patients with positive and negative C.S.A.
Assuntos
Antígenos de Helmintos/análise , Complemento C3/análise , Schistosoma/imunologia , Esquistossomose Urinária/imunologia , Esquistossomose mansoni/imunologia , Adolescente , Animais , Criança , Fezes/parasitologia , Humanos , Contagem de Ovos de ParasitasRESUMO
Transient nephritis, manifested by albuminuria may be caused by the deposition of the circulating schistosomal antigen in the kidney glomeruli.