RESUMEN
Salmonella is an important foodborne pathogen causing major public health problems throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division (gidA) gene significantly altered Salmonella virulence in both in vitro and in vivo models of infection. In Escherichia coli, GidA and MnmE have been shown to modify several bacterial factors by a post-transcriptional mechanism to modify tRNA. Therefore, we hypothesize that GidA and MnmE complex together to modulate virulence genes in Salmonella using a similar mechanism. To test our hypothesis, and to examine the relative contribution of GidA and MnmE in modulation of Salmonella virulence, we constructed gidA and mnmE single mutants as well as a gidA mnmE double mutant strain of Salmonella. Results from the in vitro data displayed a reduction in growth, motility, intracellular replication, and invasion of T84 intestinal epithelial cells in the mutant strains compared to the wild-type Salmonella strain. The in vivo data showed a significant attenuation of the mutant strains as indicated by the induction of inflammatory cytokines and chemokines, as well as in the severity of histopathological lesions in the liver and spleen, compared to mice infected with the wild-type strain. Also, a significant increase in the LD50 was observed in mice infected with the mutant strains, and mice immunized with the mutants were protected against a lethal dose of wild-type Salmonella. A pull-down assay indicated that Salmonella GidA and MnmE bind together, and HPLC analysis revealed that deletion of gidA and/or mnmE altered Salmonella tRNA modification. Overall, the data suggest MnmE and GidA bind together and use a post-transcriptional mechanism to modify tRNA to regulate Salmonella pathogenesis.
Asunto(s)
Proteínas Bacterianas/genética , GTP Fosfohidrolasas/genética , Eliminación de Gen , Salmonella/genética , Salmonella/patogenicidad , Animales , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Femenino , GTP Fosfohidrolasas/metabolismo , Humanos , Ratones , Mutación , Unión Proteica , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Salmonella/crecimiento & desarrollo , Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , VirulenciaRESUMEN
Chronic wasting disease (CWD) is a highly contagious always fatal neurodegenerative disease that is currently known to naturally infect only species of the deer family, Cervidae. CWD epidemics are occurring in free-ranging cervids at several locations in North America, and other wildlife species are certainly being exposed to infectious material. To assess the potential for transmission, we intracerebrally inoculated four species of epidemic-sympatric rodents with CWD. Transmission was efficient in all species; the onset of disease was faster in the two vole species than the two Peromyscus spp. The results for inocula prepared from CWD-positive deer with or without CWD-resistant genotypes were similar. Survival times were substantially shortened upon second passage, demonstrating adaptation. Unlike all other known prion protein sequences for cricetid rodents that possess asparagine at position 170, our red-backed voles expressed serine and refute previous suggestions that a serine in this position substantially reduces susceptibility to CWD. Given the scavenging habits of these rodent species, the apparent persistence of CWD prions in the environment, and the inevitable exposure of these rodents to CWD prions, our intracerebral challenge results indicate that further investigation of the possibility of natural transmission is warranted.
Asunto(s)
Susceptibilidad a Enfermedades/epidemiología , Enfermedad Debilitante Crónica/transmisión , Secuencia de Aminoácidos , Animales , Arvicolinae , Ciervos , Brotes de Enfermedades , Genotipo , América del Norte , Priones/genética , Roedores , Especificidad de la Especie , Enfermedad Debilitante Crónica/epidemiologíaRESUMEN
Salmonella enterica serovar Typhimurium is a frequent cause of enteric disease due to the consumption of contaminated food. Identification and characterization of bacterial factors involved in Salmonella pathogenesis would help develop effective strategies for controlling salmonellosis. To investigate the role of glucose-inhibited division gene (gidA) in Salmonella virulence, we constructed a Salmonella mutant strain in which gidA was deleted. Deletion of gidA rendered Salmonella deficient in the invasion of intestinal epithelial cells, bacterial motility, intracellular survival, and induction of cytotoxicity in host cells. Deletion of gidA rendered the organism to display a filamentous morphology compared to the normal rod-shaped nature of Salmonella. Furthermore, a significant attenuation in the induction of inflammatory cytokines and chemokines, histopathological lesions, and systemic infection was observed in mice infected with the gidA mutant. Most importantly, a significant increase in LD(50) was observed in mice infected with the gidA mutant, and mice immunized with the gidA mutant were able to survive a lethal dose of wild-type Salmonella. Additionally, deletion of gidA significantly altered the expression of several bacterial factors associated with pathogenesis as indicated by global transcriptional and proteomic profiling. Taken together, our data indicate GidA as a potential regulator of Salmonella virulence genes.
Asunto(s)
Proteínas Bacterianas/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Animales , Femenino , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Glucosa/administración & dosificación , Glucosa/metabolismo , Mucosa Intestinal/patología , Ratones , Mutagénesis Insercional , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Salmonella/microbiología , Salmonelosis Animal/microbiología , Salmonella typhimurium/citología , Salmonella typhimurium/metabolismo , Virulencia/genéticaRESUMEN
Previous work by the present authors indicated a murein lipoprotein mutant of Salmonella shows a marked down-regulation in expression of yqhC. Because YqhC is a putative DNA-binding protein, it is likely involved in modulation of Salmonella genes. Deletion of yqhC renders Salmonella defective in invasion of intestinal epithelial cells, motility, and induction of cytotoxicity. In the present study, further attenuation in induction of inflammatory cytokines/chemokines and histopathological lesions was seen in mice infected with the yqhC mutant. On the other hand, deletion of yqhC did not significantly affect the LD(50) in mice or the ability of Salmonella to survive and replicate in vivo. To better understand how YqhC affects Salmonella virulence and to identify factors potentially modulated by YqhC, comparative transcriptome and proteome analysis of the yqhC mutant and the WT Salmonella was performed. Data from these experiments indicate that deletion of yqhC significantly alters the transcription of several genes associated with the SPI-1 encoded T3SS and flagellar regulons, correlating with the yqhC mutant phenotype. Overall, this study indicates that deletion of the yqhC gene causes a number of virulence-related defects in vitro, but has a modest effect in vivo, despite affecting induction of inflammatory cytokines and histopathology.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica/genética , Prueba de Complementación Genética , Hígado/microbiología , Ratones , Viabilidad Microbiana , Fenotipo , Proteoma , Infecciones por Salmonella/mortalidad , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Eliminación de Secuencia , Bazo/microbiología , Transactivadores/genética , Transactivadores/metabolismo , Transcriptoma , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Chronic wasting disease (CWD) in Wisconsin was first identified in February 2002. By April 2005, medial retropharyngeal lymph node (RLN) tissues had been examined from over 75,000 white-tailed deer for the presence of CWD by either immunohistochemical (IHC) staining for the prion protein associated with CWD (PrP(res)) or by using enzyme-linked immunosorbent assays with confirmation of positives by IHC staining and had been detected in 469 animals. Obex tissue was also available from 438 of the CWD-positive animals and was CWD positive by IHC staining in 355 (81%). To verify whether false-negative results were possible examining only RLN, both obex and RLN samples were examined for CWD by IHC staining from 4,430 of the white-tailed deer harvested from an area in Wisconsin where the overall deer CWD prevalence was approximately 6.2%. Two hundred and fourteen of the 269 positive deer (79.6%) had deposits of PrP(res) in both obex and lymphoid tissues, 55 (20.4%) had deposits only in lymphoid tissue, and there were no deer that had deposits only in obex.
Asunto(s)
Tronco Encefálico/patología , Ciervos , Ganglios Linfáticos/patología , Enfermedad Debilitante Crónica/patología , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunohistoquímica/veterinaria , Enfermedad Debilitante Crónica/diagnósticoRESUMEN
In September 2002, chronic wasting disease (CWD), a prion disorder of captive and wild cervids, was diagnosed in a white-tailed deer (Odocoileus virginianus) from a captive farm in Wisconsin. The facility was subsequently quarantined, and in January 2006 the remaining 76 deer were depopulated. Sixty animals (79%) were found to be positive by immunohistochemical staining for the abnormal prion protein (PrP(CWD)) in at least one tissue; the prevalence of positive staining was high even in young deer. Although none of the deer displayed clinical signs suggestive of CWD at depopulation, 49 deer had considerable accumulation of the abnormal prion in the medulla at the level of the obex. Extraneural accumulation of the abnormal protein was observed in 59 deer, with accumulation in the retropharyngeal lymph node in 58 of 59 (98%), in the tonsil in 56 of 59 (95%), and in the rectal mucosal lymphoid tissue in 48 of 58 (83%). The retina was positive in 4 deer, all with marked accumulation of prion in the obex. One deer was considered positive for PrP(CWD) in the brain but not in the extraneural tissue, a novel observation in white-tailed deer. The infection rate in captive deer was 20-fold higher than in wild deer. Although weakly related to infection rates in extraneural tissues, prion genotype was strongly linked to progression of prion accumulation in the obex. Antemortem testing by biopsy of recto-anal mucosal-associated lymphoid tissue (or other peripheral lymphoid tissue) may be a useful adjunct to tonsil biopsy for surveillance in captive herds at risk for CWD infection.
Asunto(s)
Ciervos , Enfermedades por Prión/veterinaria , Enfermedad Debilitante Crónica/epidemiología , Animales , Enfermedades por Prión/epidemiología , Enfermedades por Prión/patología , Rumiantes , Enfermedad Debilitante Crónica/patología , Wisconsin/epidemiologíaRESUMEN
Accurate detection of West Nile virus (WNV) in corvids is essential for monitoring the spread of virus during the mosquito season. Viremia in corvids is very high, with titers approaching 10(8) viral particles/ml. In the presence of such marked viremia, the sensitivity of real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis is unnecessary, and more cost-effective methods should be assessed. To this end, antigen-capture ELISA (ACE) and immunohistochemical (IHC) assays were evaluated. Skin, cloacal swab specimens, and feathers from corvids were tested by use of ACE, and results were compared with results obtained from use of real-time RT-PCR analysis. Of the 3 sample types, skin gave the best sensitivity (98%) and specificity (100%). Skin, brain, kidney, and spleen from corvids were analyzed by IHC, and results were compared with real-time RT-PCR results. Kidney and spleen were more often positive by use of IHC than were brain and skin tissue; however, IHC did not perform as well as ACE in the identification of virus-positive birds. Results of this study support the use of a skin sample in an ACE format as an effective surveillance method for corvids.
Asunto(s)
Enfermedades de las Aves/diagnóstico , Cuervos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Inmunohistoquímica/veterinaria , Fiebre del Nilo Occidental/veterinaria , Virus del Nilo Occidental/aislamiento & purificación , Animales , Antígenos Virales/análisis , Enfermedades de las Aves/virología , Encéfalo/virología , Cloaca/virología , Análisis Costo-Beneficio , Ensayo de Inmunoadsorción Enzimática/economía , Ensayo de Inmunoadsorción Enzimática/métodos , Plumas/virología , Inmunohistoquímica/economía , Inmunohistoquímica/métodos , Riñón/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/economía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Piel/virología , Bazo/virología , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunologíaRESUMEN
PURPOSE: The objective of this investigation was to determine the biologic basis and the significance of uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) using experimentally created fungal lesions in rats. PROCEDURES: Uptake of FDG by experimentally induced Blastomyces granulomas was compared with uptake by turpentine abscesses (Group 1) and by lymphomas (Group 2) using the differential uptake ratio (DUR) measured one hour after administration of 2 mCi FDG intravenously. Frozen tissue sections of Blastomyces lesions and turpentine abscesses were placed in contact with radiographic film for macroautoradiography. RESULTS: In rats in Group 1, the median (range) DUR for the Blastomyces granulomas was 1.9 (1.1-2.6) and was significantly higher than the DUR for turpentine abscesses 0.9 (0.6-1.4) and muscle 0.2 (0.1-0.5; P < 0.001). In Group 2, the median (range) DUR for the Blastomyces granulomas, lymphomas, and muscle from the rats in Group 2 were 1.8 (1.2-3.4), 1.9 (1.0-4.0), and 0.2 (0.1-0.3), respectively. There was no significant difference between the DUR of Blastomyces granulomas and lymphomas. Macroautoradiographs of the Blastomyces granulomas revealed intense uptake of FDG in the region occupied by the yeast organisms and the granulomatous inflammation. CONCLUSIONS: Blastomyces granulomas typically have high uptake of FDG associated with the region composed of the granulomatous inflammatory reaction and Blastomyces yeast organisms.
RESUMEN
PURPOSE: Based on limited reports, fungal lesions can have remarkably high intensity uptake of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) on positron emission tomography (PET) images. The purpose of this investigation was to compare the standardized uptake value (SUV) of naturally occurring lesions of blastomycosis with the SUV of naturally occurring lymphoma in a series of dogs. PROCEDURES: Five dogs with naturally occurring blastomycosis and three dogs with lymphoma underwent whole-body FDG-PET prior to receiving any treatment for their disease. RESULTS: The (mean +/- SD) SUV for 13 blastomycosis lesions was 7.7 +/- 2.0 versus a mean for 17 lymphomas of 4.8 +/- 1.8. These values were significantly different (P = 0.0537). There was overlap between the SUV of Blastomyces-associated lesions versus lymphomas, but a cut-off SUV of 7.0 was 100% specific for Blastomyces lesions. Numerous sites of disease were detected on the FDG-PET images that were not detected clinically. CONCLUSIONS: FDG-PET is useful for determining the extent of disease in dogs with blastomycosis. The SUV for Blastomyces-associated lesions are as high or higher than for malignant lymphoma. Due to the similarities in canine and human blastomycosis and lymphomas, similar results would be predicted in human patients. In regions where blastomycosis is endemic, Blastomyces granulomas should be considered a differential diagnosis for lesions with high intensity uptake of FDG.
RESUMEN
OBJECTIVE: To compare prevalence of organisms and histologic changes in eyes from dogs with blastomycosis that were either untreated or undergoing treatment with itraconazole. DESIGN: Retrospective study. ANIMALS: 36 dogs with endophthalmitis associated with blastomycosis. PROCEDURE: Signalment, results of ophthalmic examination, and duration of treatment with itraconazole were extracted from medical records. Histologic sections from eyes were examined for prevalence and viability (ie, budding) of fungal organisms. A scoring system was devised to assess the degree of inflammation. RESULTS: Clinically, all eyes were blind and had signs of severe endophthalmitis. Histologically, the type and degree of inflammation and prevalence of Blastomyces dermatitidis were not significantly different between dogs treated with itraconazole and untreated dogs or among groups of dogs treated for different time periods (4 to 14, 15 to 28, or 29 to 72 days). Replication of the organisms in vascular tissues as well as avascular spaces in the eyes was similar in treated and untreated dogs. Lens rupture was seen in 12 of 29 (41%) eyes. CONCLUSIONS AND CLINICAL RELEVANCE: Persistence of inflammation in eyes of dogs with naturally occurring blastomycosis is likely attributable to the continued presence of B. dermatitidis, regardless of the duration of treatment with itraconazole. Lens capsule rupture, a common and previously unreported histologic finding, may contribute to cataract formation and continued inflammation.
Asunto(s)
Antifúngicos/uso terapéutico , Blastomicosis/veterinaria , Enfermedades de los Perros/patología , Endoftalmitis/veterinaria , Itraconazol/uso terapéutico , Animales , Blastomyces/aislamiento & purificación , Blastomicosis/tratamiento farmacológico , Blastomicosis/microbiología , Blastomicosis/patología , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/microbiología , Perros , Endoftalmitis/tratamiento farmacológico , Endoftalmitis/microbiología , Endoftalmitis/patología , Femenino , Cristalino/lesiones , Cristalino/microbiología , Masculino , Estudios RetrospectivosRESUMEN
Our purpose was to develop a simple, reliable method for creating subcutaneous Blastomyces dermatitidis nodules in rats and to describe the histologic appearance of these lesions. We used B. dermatitidis isolated from a dog with blastomycosis to prepare a Blastomyces yeast suspension. Four rats were used to test initial dose concentrations of 10(5), 10(6), 10(9), and 10(10) yeast organisms. The dose was administered subcutaneously over the distal tibia in a volume of 0.1 ml. We then inoculated 35 additional rats with 10(9) or 10(10) yeast organisms. Rats were euthanized 7, 10, 14, 21, or 28 days after inoculation, and the histologic appearance of the nodules was described. A full post-mortem examination sought evidence of systemic spread of Blastomyces organisms. We successfully induced subcutaneous Blastomyces abscesses in 34 of 37 rats injected with 10(9) or 10(10) organisms. Nodules first appeared 3 to 7 days after injection and reached 2 to 15 mm in diameter by 7 to 28 days after inoculation. Histologically the lesions were characterized by a necrotic center surrounded by a layer of viable yeast and granulomatous inflammation. Live yeast organisms were recovered from all lesions. No adverse effects or systemic spread of Blastomyces organisms were observed. We conclude that subcutaneous Blastomyces abscesses can be induced safely and reliably in rats after injection of 10(9) and 10(10) organisms. Histologically, the experimentally induced lesions share both similarities to and differences with lesions of naturally occurring blastomycosis.