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PubMed2000

Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides.

The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains. Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed. Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer. T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined. For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants. Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified. Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein. Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice. Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions. Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E. coli OmpF may have broader application.

Amino Acid Sequence↗

PubMed2003

[A simple method to identify pathogenic fungi in Sabouraud's glucose agar slant culture using fungi-tape and MycoPerm-Blue].

Pathogenic fungi can be identified by growth in special culture medium, colony appearance based on microscopic examination of conidia and hyphae, and by molecular biological techniques. However, these methods are time-consuming and labor-intensive. We describe a simple method for identification of pathogenic fungi from Sabouraud's glucose agar slant culture, using Fungi-Tape and MycoPerm-Blue. It is rapid and easy, and permits visualization of fungal morphology. Using the Fungi-Tape and MycoPerm-Blue technique, we correctly identified 47 of 66 samples, for a 73.4% concordance with conventional identification methods. It is also noteworthy that slides prepared using this method can be stored for long periods without deterioration.

Culture Media↗

PubMed1986

Future application of research tests to the diagnosis of yaws and other endemic treponematoses.

Recent advances in treponemal research have increased our understanding of the pathogenesis and host response to treponemal infection was well as the antigenic structure of the pathogenic and nonpathogenic treponemes. Although these new methodologies are currently limited to research laboratories, the new findings may ultimately have practical applications to the control of the treponematoses. It is reasonable to expect that reagents for the identification of pathogenic treponemes (vs. non-pathogens) will be commercially available in the next three years. The field application of these reagents may be limited and specimens may need to be sent to regional or reference laboratories for evaluation. Similarly, the detection of specific treponemal IgM antibody may be limited to regional or reference laboratories. This does not preclude the utility of these tests, however, in epidemiologic surveys for determination of disease prevalence before and after treatment programmes, such surveys comprise the heart of any disease control programme.

Humans↗

PubMed1989

[Bacterial infection and rapid laboratory microbial methods].

The key to treatment of bacterial infectious diseases is always to quickly identify the causative organism and understand its resistance to drugs. Recent progress in microbial laboratory methods has permitted rapid detection and identification of pathogenic organisms. Rapid test methods are classified into culture and non-culture methods. Non-culture methods are based mainly on immunoassay for detection of antigen or antibody of pathogenic organisms, but also include the DNA probe method and the RNA probe method. Immunoassay is achieved by fluorescent antibody techniques, agglutination and ELISA. On a related note, monoclonal antibodies have been developed with steady progress. For culture methods, commercial bacterial identification kits and automated instruments, all of which permit quicker identification than with conventional methods, are now used in a large number of laboratories. Quick identification with these automated instruments is possible owing to optical determination of drug resistance. Some automated instruments are capable of rapidly detecting bacteria in the sample. For example, Bactec, used for quick diagnosis of bacteremia, measures CO2 produced by bacteria in the culture bottle during the metabolic process, permitting early detection of bacterial proliferation. Other methods are available in which ATP produced by bacteria is measured on the basis of bioluminescence or chemiluminescence.

Bacteria↗

PubMed1995

Future of biotechnology-based control of disease in marine invertebrates.

Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture. Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans. Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases. The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes. This offers the opportunity for prophylactic measures to be taken. Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available. Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection. Such diagnostic tools are relatively new to aquaculture, but have enormous potential. A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens. Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp. Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes. Other transformation systems also show promise, including transposable elements and densoviruses.

Animal Diseases↗

PubMed1980

Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp.

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.

Amoeba↗

PubMed1988

Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids.

A rapid, reverse-phase high-performance liquid chromatography method was used to detect rho-bromophenacyl mycolic acid ester patterns for strains of four major pathogenic Mycobacterium species and for the most commonly encountered saprophytic species, Mycobacterium gordonae. Mycobacteria in low numbers (2.5 X 10(6) CFU) were detected and identified to the species level. Standard chromatographic patterns characteristic of each species were established. Simple pattern recognition enabled rapid identification of M. tuberculosis, M. kansasii, M. avium, M. intracellulare, and M. gordonae.

Acetophenones↗

PubMed1999

Mitochondrial DNA analysis: polymorphisms and pathogenicity.

The investigation of mtDNA disease can be relatively straightforward if a person has a recognisable phenotype and if it is possible to identify a known pathogenic mtDNA mutation. The difficulties arise when no known mtDNA defect can be found, or when the clinical abnormalities are complex and not easily matched to those of the more common mitochondrial disorders. We will describe here the difficulties that can be encountered during the identification of pathogenic mtDNA mutations and the approaches that can be used to confirm, or eliminate, a likely pathogenic role, in either single gene diseases or in multifactorial disorders.

Animals↗

PubMed1999

Evaluation of Statens Serum Institut enteric medium for detection of enteric pathogens.

The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three Vibrio spp. and one Aeromonas spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of Vibrio spp. and some Shigella spp. were required for detection. The growth rates of 125 enteric pathogens and 12 Yersinia spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12 Yersinia spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect Yersinia spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis.

Culture Media↗

PubMed2000

Expression and characterization of protein geranylgeranyltransferase type I from the pathogenic yeast Candida albicans and identification of yeast selective enzyme inhibitors.

Protein geranylgeranyltransferase type I (GGTase I) is a heterodimeric zinc metalloenzyme catalyzing protein geranylgeranylation at cysteine residues present in C-terminal signature sequences referred to as CaaX (X=Leu) motifs. We have studied GGTase I as a potential antifungal target and recently reported its purification and cloning from the yeast Candida albicans (Ca GGTase I), an important human pathogen. Here, we report the high yield bacterial expression of Ca GGTase I by coexpression of maltose binding protein fusion proteins of both the alpha (Ram2p) and beta (Cdc43p) subunits. The cleaved and purified recombinant Ca GGTase I was demonstrated to be functional and structurally intact as judged by the presence of one equivalent of a tightly bound zinc atom and the near stoichiometric formation, isolation and catalytic turnover of a geranylgeranyl pyrophosphate-GGTase I complex. Kinetic analysis was performed with a native substrate protein, Candida Cdc42p, which exhibited significant pH dependent substrate inhibition, a feature not observed with other Ca GGTase I substrates. Prenyl acceptor substrate specificity was studied with a series of peptides in which both the CaaX motif, and the sequence preceding it, were varied. The prenyl acceptor K(M)s were found to vary nearly 100-fold, with biotinyl-TRERKKKKKCVIL, modeled after a presumably geranylgeranylated Candida protein, Crl1p (Rho4p), being the optimal substrate. A screen for inhibitors of Ca GGTase I identified compounds showing selectivity for the Candida versus human GGTase I. The most potent and selective compound, L-689230, had an IC(50) of 20 nM and >12,500-fold selectivity for Ca GGTase I. The lack of significant anti-Candida activity for any of these inhibitors is consistent with the recent finding that GGTase I is not required for C. albicans viability [R. Kelly et al., J. Bacteriol. 182 (2000) 704-713].

Alkyl and Aryl Transferases↗

PubMed1991

Isolation and identification of Bacillus sphaericus strains pathogenic for mosquito larvae.

Three selective media for the isolation of Bacillus sphaericus have been compared. BATS medium and a formulation employing adenosine as the principal carbon source were the most effective for the recovery of spores of strain 1593. Anthranilic acid as the principal carbon source was less efficient. Eighty-four strains were isolated from mud samples using these media and were identified by computer. Identifications were confirmed for representative strains using DNA sequence homology. Most were B. sphaericus sensu stricto or members of an unnamed group. However, one strain (BSE 18) was identified as the DNA homology group IIB and this organism was found to be highly toxic toward larvae of Culex pipiens. Southern hybridization of BSE 18 DNA to a probe prepared from the cloned toxin gene from strain 1593 revealed that BSE 18 contained a typical gene for the 41.9-kDa toxin.

Animals↗

PubMed2000

The diagnostic value of electron microscopy in human immunodeficiency virus-positive patients with gastrointestinal disease.

BACKGROUND: Our aim was to determine the diagnostic value of electron microscopy in evaluating the etiology of gastrointestinal disease in patients infected with the human immunodeficiency virus (HIV). METHODS: A retrospective review of electron microscopic and light microscopic results of all HIV-positive patients with gastrointestinal and liver diseases was made during a 3-year period from June 1995 to June 1998. RESULTS: A total of 145 HIV-positive patients had their electron microscopy specimens reviewed. Of these, 136 were investigated for diarrhea, and the other 9 for increased liver enzymes. Twenty-seven of the 145 (18.6%) HIV-positive patients had a pathogen identified by electron microscopy, compared with only 13 of 145 (9%) identified by light microscopy (P < 0.005). The sensitivity of light microscopy for detecting opportunistic pathogens was 68%. Twenty-one of the 27 (77.8%) patients diagnosed by electron microscopy had microsporidiosis, and the most commonly diagnosed species was Enterocytozoon bieneusi. Light microscopy failed to identify 12 cases of microsporidiosis and 2 cases of leishmaniasis. CONCLUSIONS: Electron microscopy contributes substantially to the identification of pathogens in HIV-positive patients. Light microscopy failed to identify one of every two pathogens diagnosed by electron microscopy.

Adult↗

PubMedDate not supplied

[Molecular diagnosis of hereditary diseases].

Molecular biology has produced important achievements in research, and provides useful applications to the clinical field. This is true today in diagnostics, and will be true in therapeutics tomorrow. The identification of pathogenic mutations through direct analysis of known genes allows diagnoses to be reached in a growing number of disorders. When mutations cannot be identified, familial linkage studies using polymorphic molecular markers will reach a diagnosis indirectly, most of the times. This is useful either presymptomatically or prenatally. The identification of genetic risk factors in targeted populations is becoming a means of prevention of multifactorial diseases. This approach opens a very large field of applications of molecular genetics in clinical practice. Basic concepts of molecular genetics are briefly reviewed, and the principles of diagnostics in hereditary diseases are approached via a few representative examples.

Adenomatous Polyposis Coli↗

PubMed1992

Detection and identification of Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica by an improved polymerase chain reaction method.

We developed a polymerase chain reaction method in order to detect and identify both Yersinia pseudotuberculosis and pathogenic Yersinia enterocolitica. Polymerase chain reaction was performed by using a mixture of primers against the inv gene from Y. pseudotuberculosis and the ail gene from pathogenic Y. enterocolitica. Further addition of primers against the plasmid-coded virF gene from Y. enterocolitica made it possible to detect a virulence-associated gene of both species at the same time. This method was proved to be an adequate and convenient procedure for routine detection and identification of these bacilli.

Animals↗

PubMed1983

Infection in the compromised host--an overview.

The compromised host is susceptible to a wide spectrum of opportunistic pathogens. Early identification of patients who can be classified as immunocompetent as well as an understanding of the specific compromise are crucial in successful management. Since this population frequently does not exhibit overt signs and symptoms of infection, prompt action is necessary to avoid overwhelming sepsis from any number of endogenous or exogenous pathogens. A careful study of the underlying mechanisms causing increased host susceptibility as well as familiarity with some of the unusual organisms capable of causing infection assist with the medical and nursing management of the immunologically compromised host.

Age Factors↗

PubMed2004

Recent progress in the characterization of molecular determinants in the Xanthomonas axonopodis pv. manihotis-cassava interaction.

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is a widespread disease that affects cassava (Manihot esculenta Crantz). Studies on the pathogen population structure, pathogen diagnosis, identification and expression of plant genes involved in resistance have been carried out. Different molecular techniques were developed to assess the genetic diversity among the Xampopulations. Characterization of Xam population dynamics over time had enable us to determine the different factors that are associated with resistance breakdown and those that influence the genetic structure or virulence phenotypes of the pathogen's population. Methods for detecting the pathogen in vegetative planting materials and true seeds were developed and contributed to reduce the impact of the disease. To better understand the genetics of resistance a quantitative trait loci (QTLs) approach was developed. Using a PCR-based strategy with degenerate primers we isolated two resistance gene candidates in cassava. We also characterized a region of a chromosome rich in R-gene like sequence. In this review we also report the main results obtained by transcript profiling methodologies, cDNA-AFLP and ESTs developed by the authors to characterize the genes involved in disease resistance. All together these techniques allowed the identification of molecular markers either associated to CBB resistance or that may represent putative genes involved in disease resistance. This article reviews current knowledge on the molecular cassava-Xam interactions.

Chromosome Mapping↗

PubMed1998

Isolation and identification of Geotrichum candidum as an etiologic agent of geotrichosis in Bulgaria.

Geotrichosis affects mainly patients with systemic diseases like diabetes mellitus, leukoses, neoplasms etc. Clinically, it is similar to candidiasis and may occur as an oral, vaginal, skin, or systemic infection. Clinical specimens (98 sputa and 67 oral smears) were collected and studied using microscopic examination of Gram stained preparations and culture sampling between 1995 and 1997. Geotrichum candidum was isolated as a single pathogen in 8 sputum and 7 oral smear samples. Ten-day antifungal treatment with Nizoral was applied and resulted in relatively quick clinical improvement. The presented cases are the first cases of pulmonary and oral infections reported in our home practice in which Geotrichum candidum species was identified as a pathogen. The identification of Geotrichum candidum using combination of colonial and microscopic morphologic features increase the possibilities for diagnostic decision.

Bulgaria↗

PubMedDate not supplied

[Evaluation of the Yersinia enterocolitica pathogenicity through some phenotypic and genotypic characters: CRMOX agar positivity and presence of the ail gene].

Sixty-nine strains of Y. enterocolitica isolated from environmental and human matrices (waste water, food and faeces) were studied in order to evidence the presence of ail gene, calcium-dependency and Congo Red absorption for pathogenic strains identification. Out of 24 clinical strains, the ail gene was present in 21 (87%), among which 79% were CRMOX-positive as well. On the contrary, none of the 45 environmental strains showed the ail gene although only one (isolated from cooked vegetables) was CRMOX agar positive. Our results confirmed the importance of molecular methods to evidence the Y. enterocolitica pathogenic strains. However, our study pointed also the utility to consider the approach of classic bacteriology, like the subcoltivation on CRMOX agar to show calcium-dependency and Congo Red absorption. In particular, when dealing with environmental isolates, that medium will be useful as a preliminary screening to identify those isolates which need further research to indicate their pathogenic potential by the use of more complex but also more expensive molecular methods.

Agar↗