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PubMed2004

Migrational capacity of Fennoscandian populations of Venturia tremulae.

Genetic variation in three multiallelic loci was analysed with Temperature Gradient Gel Electrophoresis in order to assess the genetic population structure of Venturia tremulae var. tremulae in order to understand the evolutionary potential of the pathogen against resistance breeding. Also the identification of the fungus was verified with molecular analysis of reference isolates. The Fst and Gst values were very low indicating no substructuring or restrictions to gene flow between Fennoscandian populations of V. tremulae. The results imply high epidemiological efficiency of the fungus and therefore continuous breeding programme should be implemented for Venturia resistance of aspen.

Ascomycota↗
PubMed2001

Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae.

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5 kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.

Amino Acid Sequence↗
PubMed2002

Phenotypic characteristics and pathogenicity of Aeromonas genomospecies isolated from common carp (Cyprinus carpio L.).

AIMS: To evaluate the relationship between the genomospecies, phenotypic profile and pathogenicity for carp of 37 motile Aeromonas strains. METHODS AND RESULTS: Aeromonas strains were identified to genomospecies level by the 16S rDNA restriction fragment length polymorphism (RFLP) method and characterized phenotypically by the API 20E and API Zym systems and by conventional tube or plate methods. 16S rDNA RFLP analysis showed that the strains belonged to five species, Aeromonas bestiarum (5), Aerom. salmonicida (13), Aerom. veronii (11), Aerom. sobria (6) and Aerom. encheleia (2). Most strains of Aerom. bestiarum (80%) and Aerom. salmonicida (85%) could be separated by growth at 4 and 42 degrees C, autoagglutination after boiling, reaction for lipase (C14) and naphthol-AS-BI-phosphohydrolase. All strains of Aerom. veronii corresponded to Aerom. veronii biotype sobria and could be separated from Aerom. sobria by citrate utilization, growth at 37 and 42 degrees C, amygdalin and cellobiose fermentation. All strains of Aerom. bestiarum and most strains of Aerom. salmonicida (76.9%) and Aerom. veronii (63.6%) were pathogenic for carp. CONCLUSIONS: The biochemical identification of carp Aeromonas strains is not entirely clear. Some association between Aeromonas species, phenotypic profile and specific disease signs was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be useful for ichthyopathology laboratories in the diagnosis of motile aeromonad septicaemia in carp.

Aeromonas↗
PubMed2006

The translation of Helicobacter pylori basic research to patient care.

In 1984, Barry Marshall and Robin Warren proposed a role for bacterial infections in the pathogenesis of gastroduodenal disease, which triggered an avalanche of research intended to prove or disprove their theory. The result has been a series of advances that have enhanced our understanding of these diseases and completely modernized the clinical approach to their management. In just over 20 years, many aspects of the immunopathogenesis of these diseases have been dissected at the molecular level, with key pathogenic mechanisms being validated by the identification of genes that are associated with the development of gastric cancer. There has been particular emphasis on understanding the molecular structures associated with Helicobacter pylori and their role in modifying the host responses. Gastric immune and inflammatory responses have emerged as key elements in the pathogenesis of gastritis and epithelial cell damage. This review summarizes important findings emanating from basic research primarily related to the immunopathogenesis of H pylori that have advanced the practice of medicine or our understanding of gastroduodenal disease.

Age of Onset↗
PubMed2004

EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin.

The lethality of infection by Bacillus anthracis is largely due to its plasmid-encoded toxins, which consist of a carrier protein, the protective antigen (PA), in combination with either the lethal-factor or edema-factor moiety. During B. anthracis infections, PA secreted by bacteria binds to membrane receptors of susceptible cells, is cleaved proteolytically, attaches to lethal factor or edema factor, undergoes oligomerization and internalization, and transports its toxin partners to acidic endosomes where they are released into the cytosol. To identify specific host functions that mediate these events, we used RNA encoded by a lentivirus-based library of approximately 40,000 human ESTs to inactivate chromosomal genes in a human cell population, and we isolated clones that survived PA-dependent toxin-induced death. This phenotypic screen and subsequent analysis identified ARAP3, which is a phosphoinositide-binding protein implicated previously in membrane vesicle trafficking and cytoskeletal organization, as a mammalian host-cell gene that is essential for normal anthrax toxicity. ARAP3 deficiency produced by antisense expression of an ARAP3 EST impaired entry of PA and its bound toxigenic moieties into both human and mouse cells, resulting in reduced toxin sensitivity. Our results demonstrate the usefulness of antisense EST libraries for global chromosomal gene inactivation, establish the practicality of loss-of-function phenotypic screens for the identification of genomic loci required for pathogen effects in mammalian cells, and reveal an important role for ARAP3 in cellular internalization of anthrax toxin.

Adaptor Proteins, Signal Transducing↗
PubMed1986

Identification of sialic acids on the cell surface of hyphae and conidia of the human pathogen Fonsecaea pedrosoi.

Sialic acids were characterized on the cell surface of conidia and hyphae of Fonsecaea pedrosoi, one of the agents of chromoblastomycosis. Neuraminidase-treated conidia had a reduced negative electrophoretic mobility and, in comparison with untreated cells, bound fewer particles of colloidal iron hydroxide and of cationized ferritin. Sialic acid residues in conidia are linked to galactopyranosyl units as indicated by the increased reactivity of neuraminidase-treated cells with peanut agglutinin. N-acetylneuraminic acid was the only derivative found in the mycelium whereas conidia contained both N-glycolyl- and N-acetylneuraminic acids.

Agglutination Tests↗
PubMed2005

Hodgkin's lymphoma and tuberculosis coexistence in cervical lymph nodes.

We describe the case of a 47-year-old man admitted to the Department of Hematology because of fever, enlarged cervical and supraclavicular lymph nodes, hepatosplenomegaly and non-specific lung infiltrations. The histopathological examination of the cervical lymph node revealed Hodgkin's lymphoma (HL) NS type I. Clinical evaluation revealed stage IVB according to Ann Arbor classification and the presence of 5 unfavorable prognostic factors according to the International Prognostic Index. Despite BEACOPP chemotherapy (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone), the enlarged lymph nodes, lung infiltrations and fever persisted. Microbiological and serological tests did not lead to the identification of any viral or bacterial pathogens. Bronchoscopy showed chronic inflammation and post-tuberculosis (TB) scars in bronchi without acid-fast bacilli in bronchoalveolar lavage (BAL) in culture and polymerase chain reaction (PCR) tests. However, the biopsy of the supraclavicular lymph node revealed multiple, caseating and necrotizing granulomatous lesions with scattered Reed-Sternberg (R-S) cells. The auramin staining presented acid-fast bacilli and allowed the diagnosis of productive and caseating TB coexisting with HL. The 4 tuberculostatics regimen and ABVD chemotherapy (adriamycin, bleomycin, vincristine, dacarbazine) resulted in a complete clinical response after 3 months of treatment. In conclusion, the association between HL and TB must be considered, especially in countries where the latter is endemic. The diagnosis may be difficult due to similarities in the clinical course, laboratory tests and imaging procedures.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

Integrated minimum-set primers and unique probe design algorithms for differential detection on symptom-related pathogens.

MOTIVATION: Differential detection on symptom-related pathogens (SRP) is critical for fast identification and accurate control against epidemic diseases. Conventional polymerase chain reaction (PCR) requires a large number of unique primers to amplify selected SRP target sequences. With multiple-use primers (mu-primers), multiple targets can be amplified and detected in one PCR experiment under standard reaction condition and reduced detection complexity. However, the time complexity of designing mu-primers with the best heuristic method available is too vast. We have formulated minimum-set mu-primer design problem as a set covering problem (SCP), and used modified compact genetic algorithm (MCGA) to solve this problem optimally and efficiently. We have also proposed new strategies of primer/probe design algorithm (PDA) on combining both minimum-set (MS) mu-primers and unique (UniQ) probes. Designed primer/probe set by PDA-MS/UniQ can amplify multiple genes simultaneously upon physical presence with minimum-set mu-primer amplification (MMA) before intended differential detection with probes-array hybridization (PAH) on the selected target set of SRP. RESULTS: The proposed PDA-MS/UniQ method pursues a much smaller number of primers set compared with conventional PCR. In the simulation experiment for amplifying 12 669 target sequences, the performance of our method with 68% reduction on required mu-primers number seems to be superior to the compared heuristic approaches in both computation efficiency and reduction percentage. Our integrated PDA-MS/UniQ method is applied to the differential detection on 9 plant viruses from 4 genera with MMA and PAH of 11 mu-primers instead of 18 unique ones in conventional PCR while amplifying overall 9 target sequences. The results of wet lab experiments with integrated MMA-PAH system have successfully validated the specificity and sensitivity of the primers/probes designed with our integrated PDA-MS/UniQ method.

Algorithms↗
PubMed1994

Multi-resistance isolates possessing characteristics of both Burkholderia (Pseudomonas) cepacia and Burkholderia gladioli from patients with cystic fibrosis.

Multi-resistant strains from three UK centres, previously identified as Burkholderia (formerly Pseudomonas) cepacia, and associated with morbidity, mortality and transmission among patients with cystic fibrosis have been further characterised. Biochemical tests and fatty acid analyses indicate these strains to possess some characteristics atypical of B. cepacia but bearing close resemblance to Burkholderia gladioli, an organism previously regarded solely as a plant pathogen and a hindrance to the identification of B. cepacia. In contrast to the majority of reference strains, all multi-resistant clinical isolates possessed rough lipopolysaccharide which may be a major factor responsible for their increased antibiotic resistance and virulence. In view of the potential clinical and social problems in CF patients posed by these multi-resistant strains, it would seem prudent to consider the isolation of either B. cepacia or B. gladioli as of equal significance.

Anti-Bacterial Agents↗
PubMed2006

PHI-base: a new database for pathogen host interactions.

To utilize effectively the growing number of verified genes that mediate an organism's ability to cause disease and/or to trigger host responses, we have developed PHI-base. This is a web-accessible database that currently catalogs 405 experimentally verified pathogenicity, virulence and effector genes from 54 fungal and Oomycete pathogens, of which 176 are from animal pathogens, 227 from plant pathogens and 3 from pathogens with a fungal host. PHI-base is the first on-line resource devoted to the identification and presentation of information on fungal and Oomycete pathogenicity genes and their host interactions. As such, PHI-base is a valuable resource for the discovery of candidate targets in medically and agronomically important fungal and Oomycete pathogens for intervention with synthetic chemistries and natural products. Each entry in PHI-base is curated by domain experts and supported by strong experimental evidence (gene/transcript disruption experiments) as well as literature references in which the experiments are described. Each gene in PHI-base is presented with its nucleotide and deduced amino acid sequence as well as a detailed description of the predicted protein's function during the host infection process. To facilitate data interoperability, we have annotated genes using controlled vocabularies (Gene Ontology terms, Enzyme Commission Numbers and so on), and provide links to other external data sources (e.g. NCBI taxonomy and EMBL). We welcome new data for inclusion in PHI-base, which is freely accessed at www4.rothamsted.bbsrc.ac.uk/phibase/.

Algal Proteins↗
PubMed2006

Rational design and rapid screening of antisense oligonucleotides for prokaryotic gene modulation.

Antisense oligodeoxynucleotides (oligos) are widely used for functional studies of both prokaryotic and eukaryotic genes. However, the identification of effective target sites is a major issue in antisense applications. Here, we study a number of thermodynamic and structural parameters that may affect the potency of antisense inhibition. We develop a cell-free assay for rapid oligo screening. This assay is used for measuring the expression of Escherichia coli lacZ, the antisense target for experimental testing and validation. Based on a training set of 18 oligos, we found that structural accessibility predicted by local folding of the target mRNA is the most important predictor for antisense activity. This finding was further confirmed by a direct validation study. In this study, a set of 10 oligos was designed to target accessible sites, and another set of 10 oligos was selected to target inaccessible sites. Seven of the 10 oligos for accessible sites were found to be effective (>50% inhibition), but none of the oligos for inaccessible sites was effective. The difference in the antisense activity between the two sets of oligos was statistically significant. We also found that the predictability of antisense activity by target accessibility was greatly improved for oligos targeted to the regions upstream of the end of the active domain for beta-galactosidase, the protein encoded by lacZ. The combination of the structure-based antisense design and extension of the lacZ assay to include gene fusions will be applicable to high-throughput gene functional screening, and to the identification of new drug targets in pathogenic microbes. Design tools are available through the Sfold Web server at http://sfold.wadsworth.org.

Algorithms↗
PubMed2001

Nosocomial infections in the neonatal intensive care unit.

Nosocomial infections are significant causes of morbidity and mortality in patients who require newborn intensive care. The most common bacterial pathogens are Gram-positive bacteria, including Staphylococcus epidermidis, Staphylococcus aureus, and Enterococcus species. Gram-negative enteric bacilli and Gram-negative environmental bacteria are involved in outbreaks and occasional cases of nosocomial infection. The incidence of fungal infection has increased over the past 10 years; fungemia is the most commonly recognized infection. Surveillance for nosocomial infection is essential to identify outbreaks and detect unsuspected reservoirs of pathogens. A variety of molecular techniques can be used to determine the genetic relatedness of pathogens. Prevention of infection requires the identification of contaminated equipment, education regarding infection control methods including hand washing, and the judicious use of antimicrobial agents.

Cross Infection↗
PubMed1975

Koch's stulates and experimental ocular histoplasmosis.

The present study shows clearly that focal choroiditis is produced in rabbits by infecting the animals with a mycelial form of H. capsulatum. Identification of this organism as the pathogenic agent was made by histopathological and mycological observations. This fungus was recovered from infected ocular lesions in those eyes enucleated within four weeks following the appearance of uveitus-a time period consistent with the clinical and pathological appearance of multiple granulomas in the choroid. The absence of organisms in the contralateral eyes of these same animals at eight weeks suggests perhaps that recovery was associated with the emergence of immunity by two months following the appearance of uveitus. This is supported in part by our previous study, which supplied evidence that animals were protected from further uveitis on subsequent reinfections (after they had recovered from their initial infection) by a mycelial or yeast form of the fungus. Similar protection was also seen in animals that had prior exposures to heat-killed organisms. Moreover, onset of the ocular changes occurred usually two weeks after infection. This evidence strongly suggests that the experimental choroiditis may be immunologically induced. H. capsulatum recovered from infected eyes (Groups I and II) produced identical ocular lesions clinically and histopathologically when injected into normal animals (Groups IA and IIA). Fulfillment of Koch's postulates in experimental ocular histoplasmosis was achieved within only one month following the appearance of uveitis. This may be of fundamental importance in that efforts to demonstrate a causal relationship between the ocular picture and benign systemic histoplasmosis have been unsuccessful in man. Because of the striking similarity between the experimental choroiditis in rabbits and the changes observed in presumed ocular histoplasmosis in man, studies in primates are necessary. Since the ocular anatomy is similar in monkeys and in man, there remains the necessity to reproduce the hemorrhagic disciform lesion of the macula, which represents the gravest aspect of presumed ocular histoplasmosis.

Animals↗
PubMed2000

Noninvasive fluorescent identification of bacteria causing acute otitis media in a chinchilla model.

OBJECTIVES: To evaluate a noninvasive method of bacterial identification via fluorescence spectroscopy in the setting of acute otitis media in a chinchilla model. STUDY DESIGN: Prospective. METHODS: For each chinchilla, transbullar inoculation with Streptococcus pneumoniae, Haemophilus influenzae, or Staphylococcus aureus was performed bilaterally and clinical infection was determined by otoscopy. An optical fiber coupled to a spectrofluorometer allowed for the delivery of an excitation wavelength to the inflamed tympanic membrane and the acquisition of the resulting emission signal. Sequential emission spectra obtained over a range of excitation wavelengths were assembled by a computer algorithm, and a single, three-dimensional plot was created for each test ear. Similarly, plots from the healthy external auditory canal (EAC) were also recorded. Twelve animals were used to establish a library of four reference plots representing the three bacteria and the EAC. Of the 24 ears available for study, 10 were excluded from analysis because of lack of clinical infection or presence of tympanic membrane perforation with purulent drainage. From four additional animals, four samples from ears infected with the above bacteria and three samples of the EAC served as unknowns. The unknown plots were analyzed by an investigator blinded to their identity. RESULTS: Using a multiple correlation of the unknown to the reference plots, seven of seven samples were correctly identified. CONCLUSIONS: We were able to establish a prototype method for the noninvasive identification of a limited library of pathogens in a chinchilla model of acute otitis media.

Acute Disease↗
PubMed2000

Identification and analysis of bacterial virulence genes in vivo.

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

Animals↗
PubMed2006

In vivo correlates of infectious salmon anemia virus pathogenesis in fish.

The phenotypic correlates of pathogenicity for Infectious salmon anemia virus (ISAV) in salmonid fishes have not been thoroughly studied to date. In this study, a comparison was made of 13 different strains of ISAV, isolated from different geographical regions between 1997 and 2004, for their infectivity in three fish species [Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss)]. When the different virus isolates were used at an approximate inoculum dose of 10(6) TCID(50) in 0.2 ml per fish, it was found that the most virulent strains had an acute mortality phase in Atlantic salmon that started at 10-13 days post-inoculation and lasted for 9-15 days with a cumulative mortality of >/=90 %. These highly pathogenic strains also caused low mortality in rainbow trout, albeit later in infection. Viruses with a more delayed or protracted mortality phase resulting in cumulative mortalities of 50-89 % in Atlantic salmon were considered to be of intermediate pathogenicity and isolates with cumulative mortalities of </=49 % were considered to be of low pathogenicity. On this basis, three of the ISAV isolates showed a high-, eight an intermediate- and two a low-pathogenicity phenotype in Atlantic salmon. Coho salmon were resistant to all ISAV isolates. These results confirmed that there is variation in pathogenicity among ISAV strains for Atlantic salmon and rainbow trout, and that other salmonid species such as coho salmon can carry highly pathogenic strains of ISAV without showing signs of disease. The identified pathogenicity phenotypes may aid in the identification of molecular markers of ISAV virulence.

Anemia↗
PubMed1994

Identification and molecular characterization of a major ring-forming surface protein from the gastric pathogen Helicobacter mustelae.

The spiral microaerophilic bacterium Helicobacter mustelae is linked to gastritis and gastric ulcers in ferrets. Electron microscopy of H. mustelae showed the presence of a laterally extensive array of 8.5-nm-diameter rings on the cell surface, which was shown to be composed of a 150kDa protein. This protein was purified, and the sequence of 10 amino-terminal residues was determined. Polyclonal antibody against the purified 150 kDa protein labelled the ring structures on the homologous strain by means of immunogold. Cross-reactive proteins were identified in three H. mustelae strains, but not in Helicobacter pylori or Helicobacter felis. The hsr gene encoding this protein was cloned, and the protein expressed in Escherichia coli independently of vector promoters. The 1519-codon nucleotide sequence of the gene was determined, and comparison with the chemically derived protein sequence indicated a 47-residue leader peptide, and a mature protein with a molecular weight of 152,300. Thus the cell surface of H. mustelae differs markedly from other members of the genus Helicobacter in being covered by an array of 8.5 nm rings composed of a 150kDa protein.

Amino Acid Sequence↗
PubMed2006

Probiotics in marine larviculture.

Owing to the problem of antibiotic resistance and subsequent reluctance of using antibiotics, the use of probiotics in larviculture is becoming increasingly popular. During the early stages of development, manipulation of the larval digestive system seems possible through the addition of probiotics either through the culture water or via the livefood. Well-studied probiotics used in human medicine and terrestrial agriculture have proved to be successful in aquaculture and therefore reduce the need for extensive biosafety trials. The selection of probiotics requires various in vitro screening experiments, which assay for the production of antagonist compounds, their growth in and attachment to fish intestinal mucus, and the production of other beneficial compounds such as vitamins, fatty acids and digestive enzymes. Further information regarding probiont suitability can be obtained from its identification, interaction with livefood and host pathogenicity. Finally, pilot-scale in vivo tests need be performed, after which a production cost-benefit analysis to determine its economic viability needs to be undertaken.

Animals↗

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