[Cloning of DNA probes for detection and identification of pathogenic fungi Verticillium dahliae i Verticillium tricorpus].
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Base Sequence↗
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Results for “pathogen detection”
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[Biological features and problems in detection of extrapulmonary tuberculosis pathogen].
The viability, enzymatic activity and Mycobacterium tuberculosis drug resistance was compared in pulmonary and extrapulmonary tuberculosis. To decrease oxygen in the culture medium baseline values and to use various oxidants as protectors were shown to be promising in enhancing the efficiency of cultural diagnosis of extrapulmonary tuberculosis.
Antitubercular Agents↗
The microbiology of acute diarrhoeal disease in the eastern province of Saudi Arabia.
A prospective study of acute diarrhoeal diseases in the Eastern Province of Saudi Arabia was carried out over a 19-month period to determine aetiology, risk factors and other epidemiological characteristics. Of the 853 subjects studied, 344 were cases and 509 controls. More cases were seen in children than in adults. Enteric pathogens were detected in 49% of the cases, but none in the controls. Of the pathogens, 68% were bacterial with Salmonella (34%) and Shigella species (14.7%) being the most common; Campylobacter jejuni emerged as an important cause especially in adults. Contrary to most reports, rotaviruses were responsible for only 11.5% of the cases in children. Entamoeba histolytica (13.5%) and Giardia intestinalis (10.4%) were parasites commonly detected. Shigella organisms were the only pathogens that were isolated in hospital cases without being isolated in cases from the community.
Acute Disease↗
The 18S rDNA sequence of Synchytrium endobioticum and its utility in microarrays for the simultaneous detection of fungal and viral pathogens of potato.
Resting spores extracted from wart (Synchytrium endobioticum)-infected potato tubers were used for DNA extraction and amplification of 18S rDNA. Analysis of the cloned, sequenced fragment revealed high similarity to members of the Chytridiomycota. Using this information, specific oligonucleotide probes were designed and arrayed onto glass slides for detection of the pathogen. Viral sequence information available in the databank was retrieved, or new viral sequences were generated, and used to design probes for specific detection of important quarantine viruses of potato. To determine the sensitivity and specificity of the oligonucleotide probes, total RNA from infected plants was reverse transcribed, labelled with Cyanine 5, and hybridised with the microarray. A significant number of the oligonucleotide probes exhibited high specificity to S. endobioticum, Andean potato latent virus, Andean potato mottle virus, Potato black ringspot virus, and Potato spindle tuber viroid. Hybridisation signals of sub-arrays within slides were reproducible (r = 0.79) with a high correlation coefficient of hybridisation repetitions (0.73). Our results demonstrate the potential of microarray-based hybridisation for identification of multiple pathogen targets, which will find application in quarantine laboratories, where parallel testing for diverse pathogens is essential.
Chytridiomycota↗
Development of a fluorogenic 5' nuclease PCR assay for detection of the ail gene of pathogenic Yersinia enterocolitica.
In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect </=4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 10(8) CFU of contaminating bacteria per ml. This set was also capable of detecting </=1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.
Animals↗
Association between pathogenicity of infectious bursal disease virus and viral antigen distribution detected by immunohistochemistry.
Highly pathogenic infectious bursal disease virus (IBDV) strains (Ehime/91, DV86) and a moderately pathogenic strain (J1) were compared in order to clarify the association between the pathogenicity of IBDV and viral antigen distribution. Virus target cells in the bursa, thymus, spleen, and bone marrow were examined by transmission electron microscopy. Although all strains caused similar bursal atrophy, the highly pathogenic strains brought about a greater decrease in the thymic weight index and more severe lesions in the cecal tonsil, thymus, spleen, and bone marrow. Immunohistochemical detection of IBDV antigen in tissues from chickens infected with Ehime/91 and DV86 strains showed a higher frequency of antigen-positive cells in the spleen and bone marrow. Transmission electron microscopy indicated the presence of viral particles in the cytoplasm of epithelial reticular cells in the thymus and monocytes in the bone marrow. The results show that pathogenicity of field strains of IBDV correlates with lesion production in non-bursal lymphopoietic organs. The results also suggest that pathogenicity of IBDV may be associated with virus antigen distribution in non-bursal lymphopoietic organs.
Animals↗
Predictions for rapid methods and automation in food microbiology.
A discussion is presented on the present status of rapid methods and automation in microbiology. Predictions are also presented for development in the following areas: viable cell counts; real-time monitoring of hygiene; polymerase chain reaction, ribotyping, and genetic tests in food laboratories; automated enzyme-linked immunosorbent assay and immunotests; rapid dipstick technology; biosensors for Hazard Analysis Critical Control Point programs; instant detection of target pathogens by computer-generated matrix; effective separation and concentration for rapid identification of target cells; microbiological alert systems in food packages; and rapid alert kits for detecting pathogens at home.
Automation↗
Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents.
AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.
4-Butyrolactone↗
Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes.
We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture. The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome. Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag. The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection. Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level. Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented. The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca. 30 CFU mg(-1) of tissue was estimated. A similar detection limit was observed for several other gram-negative pathogens. This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.
Aeromonas↗
Fluorogenic substrate detection of viable intracellular and extracellular pathogenic protozoa.
Viable Leishmania promastigotes and amastigotes were detected by epifluorescence microscopy with fluorescein diacetate being used to mark living parasites and the nucleic acid-binding compound ethidium bromide to stain dead cells. This procedure is superior to other assays because it is faster and detects viable intracellular as well as extracellular Leishmania. Furthermore, destruction of intracellular pathogens by macrophages is more accurately determined with fluorescein diacetate than with other stains. The procedure may have applications in programs to develop drugs and vaccines against protozoa responsible for human and animal disease.
Animals↗
Serological monitoring on layer farms with specific pathogen-free chickens.
To monitor the existence of avian pathogens in laying chicken flocks, specific pathogen-free (SPF) chickens were introduced into two layer farms and reared with laying hens for 12 months. SPF chickens were bled several times after their introduction and examined for their sero-conversion to avian pathogens. As a result, antibodies to eight or ten kinds of pathogens were detected in SPF chickens on each farm. Antibodies to infectious bronchitis virus (IBV), avian nephritis virus, Mycoplasma gallisepticum and M. synoviae were detected early within the first month. Antibody titer to IBV suggested that the laying chickens were infected with IBV repeatedly during the experiment on both farms. However, antibodies to infectious bursal disease virus and 6 pathogens were not detected.
Animals↗
Validation of salmonellosis and shigellosis diagnostic test kits at a provincial hospital in Thailand.
Rapid Diagnosis of salmonellosis and shigellosis was performed using six different diagnostic test kits which recently have been made available commercially. They were Salmo-Dot, Typhi-Dot, Shigel Dot A, B, C, and D test kits for detection of Salmonella spp., group D salmonellae, and groups A, B, C, and D Shigella spp., respectively. The principle of all test kits is a membrane (dot) ELISA using specific monoclonal antibodies to the respective pathogens as the detection reagents. The present study was designed to validate the accuracy of the test kits, at a laboratory in a provincial hospital in Thailand, in comparison with the conventional bacterial culture method alone or with the combined results of the culture and the Western blot analysis (WB) for detecting the respective bacterial lipopolysacchharides (LPS) in specimens. Five hundred rectal swab samples of patients with diarrhea who seeked treatment at the hospital, were evaluated. The diagnostic accuracy of the Salmo-Dot was 91.0% when compared with the conventional bacterial culture method alone but was 100.0% in comparison with the combined results of the culture and the WB. The Typhi-Dot and the Shigel-Dot A, B, C, and D showed 100%, 99.2%, 95.0%, 94.0% and 96.4%, respectively when compared with the culture alone and all were 100% in comparison with the combination of the results of the bacterial culture and the WB. The Shigel-Dot A revealed antigen of type 1 Shigella dysenteriae in several specimens in which the bacteria could not be recovered by the culture method. This difference is important as type 1 Shigella dysenteriae have high epidemic potential and often cause severe morbidity. Unawareness of their presence by the conventional culture may have great impact on disease surveillance for public health. The pathogen detection using the six diagnostic test kits is sensitive, specific, rapid, and relatively simple and less expensive. Several specimens can be tested at the same time without much increase in turn around time. Moreover, these kits produce no contaminated waste as compared with the bacterial culture method. The test kits should be used for rapid screening of specimens of patients with diarrhea especially in areas where culture facilities are inadequate.
Diagnosis, Differential↗
Community-acquired pneumonia of mixed etiology: prevalence, clinical characteristics, and outcome.
Community-acquired pneumonia (CAP) of mixed etiology has increasingly been appreciated in the literature, but its clinical significance remains unknown. The aim of this analysis was to describe the prevalence, clinical characteristics, and outcome of CAP of mixed etiology. Data were obtained from a 2-year prospective study of consecutive patients with CAP in whom an extensive microbiological workup was performed. Predefined strict criteria were used to establish the etiology. A total of 493 patients were included. A single pathogen was detected in 222 (45%) cases and two or more pathogens in 28 (5.7%) cases. Mixed infections were seen across all age groups and in patients treated both in hospital and as outpatients. The most frequent combinations of pathogens were those of a bacterium plus an "atypical" organism (28.6%) and of two bacterial organisms (28.6%). Compared with patients with monomicrobial pneumonia, patients with mixed pneumonia were more likely to have underlying conditions (64% vs. 45%, p=0.04) and dementia (25% vs. 10%, p=0.02). The incidence of a defined series of complications was higher in patients with mixed pneumonia (39.3% vs. 18.6%; OR=2.84; p=0.02). Community-acquired pneumonia of mixed etiology is uncommon. Patients with mixed pneumonia are more likely to have underlying medical conditions, and they may have a more severe course of disease.
Adolescent↗
The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4.
Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns--including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA--and in turn activate effector functions, including anti-apoptotic signalling pathways. Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.
Animals↗
The effect of pH and culture system on the attachment of plasmid-bearing and plasmid-cured Yersinia enterocolitica to a polycarbonate membrane in a surface adhesion immunofluorescent technique.
A rapid surface adhesion based immunofluorescence technique was used to isolate and detect Yersinia enterocolitica from inoculated enriched culture systems. The pathogen was isolated by surface adhesion to a polycarbonate membrane which was mounted on a glass slide and immersed in the enriched culture for 15 min. The pathogen was detected using a fluorescent labelled (FITC) monoclonal antibody which was specific for Y. enterocolitica serotype O:3 and then viewed using fluorescent microscopy. The effect of culture type (broth, meat homogenate and minced meat) and pH (5.00, 7.00, 9.00 and 11.00) on the adhesion of plasmid-bearing and plasmid-cured Y. enterocolitica to the polycarbonate membrane in this technique was determined. The pH had a significant effect (P < 0.05) in broth and meat homogenate cultures, with enhanced attachment of Y. enterocolitica (P+ and P-) at pH 9.00 than at pH 5, 7 or 11. Culture type was also important, with differences observed in the numbers of Yersinia adhering to membranes immersed in broth, meat homogenate and minced beef. Differences in attachment were noted between plasmid-bearing and plasmid-cured Y. enterocolitica isolated from similar cultural environments. The reasons for these observed differences, and their implications for the surface adhesion immunofluorescent rapid method, are discussed.
Bacterial Adhesion↗
Acute community-acquired diarrhea requiring hospital admission in Swiss children.
In order to ascertain the prevalence of agents that cause childhood diarrheal illness, stool specimens of 312 consecutive children with community-acquired diarrhea requiring admission were evaluated. Pathogens were detected in 166 (53%) of the 312 children (>/=2 pathogens in 28 children): Rotavirus (n=75), Salmonella spp. (n=37), Campylobacter spp. (n=24), Shigella spp. (n=5), Giardia spp. (n=4), Yersinia spp. (n=2), Aeromonas spp. (n=15), Cryptosporidium (n=15), enteropathogenic Escherichia coli (n=13), enterotoxigenic E. coli (n=7), and enterohemorrhagic E. coli (n=5). In conclusion, acute childhood diarrheal illness pathogens, such as Aeromonas, Cryptosporidium, and diarrheagenic E. coli, account for a large proportion of patients with a microbiologically positive stool specimen.
Adolescent↗
Screening of semen donors for infectious diseases.
The aim of this work was to prepare European guidelines concerning the screening for infectious diseases in the setting of gamete donation. Existing guidelines have been reviewed and commented. In particular, the problem of cytomegalovirus (CMV) was a matter of debate following the decision by the French Government to reject CMV seropositive donors. Other remarks and suggestions concerning various pathogens and perspectives of new molecular techniques for the detection of pathogens are also included.
Antibodies, Viral↗
Identification of different bacterial DNAs in human coronary arteries.
BACKGROUND: Various studies have suggested a link between infection, atherosclerosis and coronary artery disease. We studied whether bacterial DNA is present in coronary specimens obtained from left anterior descending coronary arteries of subjects having sudden deaths of cardiovascular and other causes, as verified by an autopsy. MATERIALS AND METHODS: Coronary specimens were obtained from five subjects who died of sudden coronary causes and five controls. Broad-range 16-s rDNA PCR (Br-PCR) amplification, cloning and sequencing were used to detect bacterial rDNA. RESULTS: Bacterial rDNA sequences of oral pathogens were detected from the coronary samples in all cases regardless of the cause of death. CONCLUSIONS: Br-PCR is a powerful method to detect bacterial rDNA. By this method we were able to detect wide palette of oral bacteria from coronary tissues. Our findings suggest that atheromas may act as mechanical sieves collecting bacteria from the circulation.
Adult↗