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At least 1,621 records · Page 90Linked to original sources
PubMed2003

Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale, A. centrale, and A. ovis and the specific detection of A. centrale vaccine strain.

Phenotypic criteria for the identification of erythrocytic ruminant Anaplasma species has relied on subjective identification methods such as host pathogenicity (virulence for cattle or sheep) and/or the location of Anaplasma inclusion bodies within the host's red cells. Sequence comparisons of new and available GenBank Accessions were investigated to elucidate the relationships among these closely related Anaplasma species. Twenty-one 16S rDNA and GroEL (HSP60) sequences from 13 Anaplasma marginale (South Africa, Namibia, Zimbabwe, Israel, USA, Australia and Uruguay), three A. centrale (South Africa and Japan), two A. ovis (USA and South Africa), and two unknown Anaplasma species isolated from wild ruminants (South Africa), were compared. 16S rDNA maximum-likelihood and distance trees separated all A. marginale (and the two wild ruminant isolates) from the two South African A. centrale (including original vaccine strain, Theiler, 1911). The Japanese A. centrale (Aomori) demonstrated the lowest sequence identity to the remaining erythrocytic Anaplasma species. A. ovis inter-species relationships could not be resolved through the 16S rDNA analyses, whereas strong bootstrap branch support is demonstrated in the GroEL distance tree using A. ovis OVI strain. All erythrocytic Anaplasma species and isolates were confirmed to belong to the same cluster showing strong branch support to Anaplasma (Ehrlichia) phagocytophilum with Ehrlichia (Cowdria) ruminantium and Rickettsia rickettsii serving as appropriate out-groups. Based on groEL sequences, a specific PCR method was developed which amplified A. centrale vaccine (Theiler, 1911) specifically. This study confirms the suitability of 16S rDNA sequences to define genera and demonstrates the usefulness of GroEL sequences for defining species of erythrocytic Anaplasma.

Anaplasma↗
PubMed2002

High-throughput approaches to study salivary proteins and genes from vectors of disease.

Blood-feeding arthropods have in their saliva a variety of molecules that affect the vertebrate host's hemostatic, inflammatory, and immune systems. The saliva of blood feeders also helps to facilitate infection of the pathogens they carry to the host, making vector saliva an attractive target to control pathogen transmission. The isolation and identification of salivary molecules from vectors of disease has been slow and difficult. Emerging technologies and new approaches in the fields of molecular biology and protein chemistry are facilitating this work. Massive sequencing of high quality, full-length cDNA libraries, coupled with proteomics and functional genomic approaches has led to the discovery of novel proteins, transcription products (genes), and biologic activities from the salivary glands of blood-feeding arthropods. This review focuses on the biologic activities identified in the saliva of various vectors of disease using classic biochemical and molecular biology approaches and new types of molecules and activities identified with high-throughput strategies.

Animals↗
PubMed2000

Receptor kinase activation and signal transduction in plants: an emerging picture.

Plant receptor kinases play key roles in the cell-cell recognition process during development, defense against pathogens, and self incompatibility. Recent identification of potential ligand molecules and downstream signaling components, together with biochemical studies on receptor-complex formation, have revealed an emerging picture of receptor-kinase activation and signal transduction in plants.

Plants↗
PubMed2005

Subversion of host cell signalling by the protozoan parasite Leishmania.

The protozoa Leishmania spp. are obligate intracellular parasites that inhabit the macrophages of their host. Since macrophages are specialized for the identification and destruction of invading pathogens, both directly and by triggering an innate immune response, Leishmania have evolved a number of mechanisms for suppressing some critical macrophage activities. In this review, we discuss how various species of Leishmania distort the host macrophage's own signalling pathways to repress the expression of various cytokines and microbicidal molecules (nitric oxide and reactive oxygen species), and antigen presentation. In particular, we describe how MAP Kinase and JAK/STAT cascades are repressed, and intracellular Ca2+ and the activities of protein tyrosine phosphatases, in particular SHP-1, are elevated.

Animals↗
PubMed1998

Molecular detection of parasitic protozoa.

The development of molecular diagnostic methods, particularly those utilizing PCR for the detection of parasitic protozoa will contribute greatly to the identification and control of these pathogens, by increasing speed of diagnosis, specificity and sensitivity, reproducibility and case of interpretation. PCR methods are not without their problems however, and there is a need for laboratory procedures to be refined before PCR-based assays are accepted as the tools of choice for the routine detection of protozoan parasites. The application of PCR detection to various parasites is discussed.

Animals↗
PubMed2000

Microbiological diagnosis of invasive fungal infections in transplant recipients.

Invasive fungal infections remain an important cause of morbidity and mortality in transplant recipients. Since conventional diagnostic tools such as culture lack sensitivity and specificity, alternative diagnostic assays have been developed. Among the most promising techniques are the detection of fungal DNA and serology. Fungal DNA can be detected with high sensitivity and specificity when performed with specimens from sterile sites such as blood. Polymerase chain reaction (PCR) assays can be used to detect a broad range of fungal pathogens and combined with species identification. Multicenter diagnostic studies are needed to establish the diagnostic value of PCR but lack of standardization hampers such studies. The serodiagnosis of invasive fungal infections has become an important tool in the management of invasive fungal infections. Especially the detection of circulating galactomannan has been shown to be a sensitive marker for invasive aspergillosis. Both serology and PCR can be used to monitor the response to antifungal therapy. The optimal use of non-culture-based methods is in prospective screening of patients at high risk. Since the incidence of disease greatly influences the positive predictive value, screening should take place only in those patients at very high risk for invasive fungal disease.

Aspergillosis↗
PubMed2004

TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM.

Both plants and animals respond to infection by synthesizing compounds that directly inhibit or kill invading pathogens. We report here the identification of infection-inducible antimicrobial peptides in Caenorhabditis elegans. Expression of two of these peptides, NLP-29 and NLP-31, was differentially regulated by fungal and bacterial infection and was controlled in part by tir-1, which encodes an ortholog of SARM, a Toll-interleukin 1 receptor (TIR) domain protein. Inactivation of tir-1 by RNA interference caused increased susceptibility to infection. We identify protein partners for TIR-1 and show that the small GTPase Rab1 and the f subunit of ATP synthase participate specifically in the control of antimicrobial peptide gene expression. As the activity of tir-1 was independent of the single nematode Toll-like receptor, TIR-1 may represent a component of a previously uncharacterized, but conserved, innate immune signaling pathway.

Amino Acid Sequence↗
PubMed1997

Evaluation of the resolving power of three different DNA fingerprinting methods to discriminate among isolates of a natural Rhizobium meliloti population.

In a comparative study, the PCR-based RAPD and ERIC fingerprint methods were evaluated for their resolving power to discriminate among 21 isolates of a natural Rhizobium meliloti population. PCR fingerprint patterns were analysed by using an automated laser fluorescent (ALF) DNA sequencer, thus allowing the automated on-line storage of data. Results obtained were compared to a classification system using insertion sequence (IS) fingerprinting. Both PCR fingerprint methods were comparable in their ability to resolve differences amongst Rh. meliloti isolates. Grouping of strains on the basis of their RAPD as well as their ERIC fingerprints correlated with grouping of strains according to their IS fingerprints. Moreover, strains displaying identical PCR patterns could be further differentiated according to their IS fingerprints, thus allowing a detailed insight into phylogenetic relationship among strains. The automated evaluation of strain-specific fingerprint patterns has the potential to become a valuable tool for studies of bacterial population genetics. Moreover, the rapid identification of single strains, e.g. pathogens in epidemiological studies seems feasible.

Bacterial Typing Techniques↗
PubMed2000

Comparative proteome analysis of Helicobacter pylori.

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

Bacterial Proteins↗
PubMed2004

A previously undescribed coronavirus associated with respiratory disease in humans.

The etiology of acute respiratory tract illnesses is sometimes unclear due to limitations of diagnostic tests or the existence of as-yet-unidentified pathogens. Here we describe the identification and characterization of a not previously recognized coronavirus obtained from an 8-mo-old boy suffering from pneumonia. This coronavirus replicated efficiently in tertiary monkey kidney cells and Vero cells, in contrast to human coronaviruses (HCoV) 229E and OC43. The entire cDNA genome sequence of the previously undescribed coronavirus was determined, revealing that it is most closely related to porcine epidemic diarrhea virus and HCoV 229E. The maximum amino acid sequence identity between ORFs of the newly discovered coronavirus and related group 1 coronaviruses ranged from 43% to 67%. Real-time RT-PCR assays were designed to test for the prevalence of the previously undescribed coronavirus in humans. Using these tests, the virus was detected in four of 139 individuals (3%) who were suffering from respiratory illness with unknown etiology. All four patients suffered from fever, runny nose, and dry cough, and all four had underlying or additional morbidity. Our data will enable the development of diagnostic tests to study the prevalence and clinical impact of this virus in humans in more detail. Moreover, it will be important to discriminate this previously undescribed coronavirus from HCoV 229E and OC43 and the severe acute respiratory syndrome coronavirus.

Animals↗
PubMed2006

Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens.

We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target ('tester'). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens ('driver'). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies.

Caliciviridae↗
PubMed2004

Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage.

Different biotic stresses yield specific symptoms, owing to their distinct influence on a plant's physiological status. To monitor early changes in a plant's physiological status upon pathogen attack, chlorophyll fluorescence imaging (Chl-FI) and thermography, which respectively visualize photosynthetic efficiency and transpiration, were carried out in parallel for two fundamentally different plant-pathogen interactions. These non-destructive imaging techniques were able to visualize infections at an early stage, before damage appeared. Under growth-room conditions, a robotized set-up captured time series of visual, thermal and chlorophyll fluorescence images from infected regions on attached leaves. As a first symptom of the plant-virus interaction between resistant tobacco and tobacco mosaic virus (TMV), thermal imaging detected a local rise in temperature while Chl-FI monitored a co-localized increase in fluorescence intensity. Chl-FI also revealed pre-symptomatic high-intensity spots for the plant-fungus system sugar beet-Cercospora beticola. Concomitantly, spots of lower temperature were monitored with thermography, in marked contrast with our observations on TMV-infection in tobacco. Knowledge of disease signatures for different plant-pathogen interactions could allow early identification of emerging biotic stresses in crops, facilitating the containment of disease outbreaks. Presymptomatic monitoring clearly opens perspectives for quantitative screening for disease resistance, either on excised leaf pieces or attached leaves.

Ascomycota↗
PubMed2003

High percentages of resistance to tetracycline and penicillin and reduced susceptibility to azithromycin characterize the majority of strain types of Neisseria gonorrhoeae isolates in Cuba, 1995-1998.

BACKGROUND: In many regions the susceptibility of Neisseria gonorrhoeae isolates to antimicrobial agents is rarely tested. The Gonococcal Antimicrobial Surveillance Program (GASP) in Cuba was established as part of a larger regional GASP program to facilitate the collection and reporting of antimicrobial susceptibility data for N gonorrhoeae isolates. GOAL: The goal was to retrospectively determine the antimicrobial susceptibility and molecular epidemiology of 91 isolates of N gonorrhoeae isolated from 11 centers in Cuba. STUDY DESIGN: Isolates of N gonorrhoeae were collected and presumptively identified from 11 Cuban provincial health centers. They were then forwarded to the National Laboratory of Pathogenic Neisseria Havana for confirmatory identification and were subsequently analyzed at the Center for GASP in Ottawa. Isolates were tested for susceptibility to penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, and azithromycin by the agar dilution method. To establish baseline data for molecular epidemiologic profiles, the auxotype (A), serovar (S), plasmid content (P), and TetM type of the isolates were determined. Certain A/S/P classes were further analyzed by pulsed field gel electrophoresis (PFGE). RESULTS: High percentages of the 91 N gonorrhoeae isolates were resistant to penicillin (68%) and tetracycline (83.5%), with 56% being penicillinase-producing (PPNG) and 64% carrying plasmid-mediated tetracycline resistance (TRNG; 50% were PP/TRNG). An additional 14% of the isolates carried chromosomal resistance (CMRNG) to either tetracycline or penicillin or both antibiotics. All isolates were susceptible to spectinomycin, ceftriaxone, and ciprofloxacin. However, nine isolates were resistant to azithromycin (MIC, > or = 1.0 microgram/ml), and 43 other isolates displayed reduced susceptibility to this antibiotic (MIC, 0.25-0.5 microgram/ml). Although a total of 21 different A/S classes were identified, most of the isolates (61) belonged to three A/S classes: NR/IA-6 (35 isolates), NR/IB-1 (15 isolates), and P/IA-6 (11 isolates). Thirty-two of 45 PP/TRNG were A/S class NR/IA-6, and nine of the P/IA-6 isolates were TRNG. By contrast, most of A/S class NR/IB-1 (8) were CMRNG. PFGE analysis following digestion with NheI or SpeI further clustered the isolates into separate groups. CONCLUSIONS: This study demonstrates high percentages of N gonorrhoeae isolates with penicillin and tetracycline resistance in Cuba. As has been noted in other studies in the Caribbean region and Latin America, resistance and reduced susceptibility to azithromycin are developing as emerging problems. Since penicillin and tetracycline continue to be widely used for the treatment of gonococcal infections in Cuba, this study indicates the importance of antimicrobial susceptibility surveillance so that effective antibiotics may be recommended for treatment of gonococcal infections.

Anti-Bacterial Agents↗
PubMed2006

An analysis of pediatric blood cultures in the postpneumococcal conjugate vaccine era in a community hospital emergency department.

OBJECTIVE: Blood cultures are commonly included in the evaluation of febrile children younger than 3 years without focal source of infection. Clinicians treat patients with a positive blood culture before final identification of the organism. Their treatment might include reevaluation in the emergency department (ED), additional tests, parenteral antibiotics, and hospital admission even for children who ultimately have false-positive (FP) blood cultures. The advent of pneumococcal conjugate vaccine (PCV) has made occult bacteremia less common, decreasing the likelihood that a positive blood culture result before final organism identification will be a true pathogen. This study will identify the characteristics of patients with FP blood cultures in the post-PCV era. METHODS: Charts were reviewed of all children ages 1 to 36 months with a temperature of at least 38.08 degrees C who had a blood culture obtained in our community hospital ED from January 1997 to January 2005. RESULTS: Bacteria grew in 106 (3.5%) out of 2971 blood cultures. True positives (TPs), defined as true pathogens, had a prevalence of 0.7%, representing 19.8% of positives. FPs, defined as contaminants, occurred in 2.8% of cultures, representing 80.2% of positives. Patients with FP cultures had lower mean white blood cell (WBC) counts (10.51 x 10(9)/L vs. 16.95 x 10(9)/L; P = 0.0001) and lower mean presenting temperatures (38.8 degrees C vs. 39.4 degrees C; P = 0.005). FPs had longer time to positivity (34.6 vs. 17.7 hours; P = 0.001) than TPs. A culture with a Gram stain suggestive of a contaminant, time to positivity greater than 24 hours and a WBC of less than 15 x 10(9)/L had a PPV for an FP of 97%. When analysis was restricted to well-appearing children age 2 to 36 months with temperature of more than 39 degrees C without focal source of infection who were discharged from the ED, these three criteria had a PPV for an FP of 100%. In these highly febrile children, the FPs had significantly lower WBCs (9.14 x 10(9)/L vs. 22.84 x 10(9)/L; P = 0.0001) and longer time topositivity (33.4 vs. 19.8 hours; P = 0.007) than TPs. The likelihood of obtaining FP cultures increased after the introduction of PCV from 62.5% to 87.8% odds ratio, 4.3; 95%confidence internal, 1.44-13.38). CONCLUSIONS: In the post-PCV era, the majority of blood culture results will be FPs. FP cultures are predictable in febrile children with WBC counts less than 15.00 x 10(9)/L, time to positivity of more than 24 hours, and a Gram stain result suggestive of a contaminant. Prospective studies applying these criteria to the at-risk population for occult bacteremia are indicated.

Bacteremia↗
PubMed2006

Genetic susceptibility to neonatal infection.

PURPOSE OF REVIEW: To review current data on genetic factors contributing to the striking susceptibility of neonates to infectious diseases and other adverse outcomes. RECENT FINDINGS: Although few studies address genetic determinants of neonatal infectious disease susceptibility, several variants in genes involved in the innate immune response have been associated with differential risk for neonatal infection. The most consistent results relate to polymorphisms of tumour necrosis factor-alpha, whereas other gene polymorphisms, such as those of interleukin-6, have yielded conflicting findings. Similar genetic factors may be involved in other inflammatory neonatal diseases. Recent data suggest that genetic variation may influence the pace of immunologic maturation. SUMMARY: Despite the enormous human and financial costs of infection for neonatal mortality and morbidity worldwide, it remains unclear why neonates are so susceptible. Genetic epidemiologic studies may assist in the identification of critical protective and pathogenic pathways. Despite the current relative lack of robust data, such studies are likely to facilitate the development of interventions that ultimately decrease the significant morbidity and mortality of this highly vulnerable population.

Communicable Diseases↗
PubMed2005

Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray.

Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.

Agriculture↗
PubMed2000

Analysis of Leptospira isolates from mainland Portugal and the Azores islands.

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.

Animals↗
PubMed1991

Recent advances in the polymerase chain reaction.

The polymerase chain reaction (PCR) has dramatically altered how molecular studies are conducted as well as what questions can be asked. In addition to simplifying molecular tasks typically carried out with the use of recombinant DNA technology, PCR has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences. PCR can provide insights into the intricacies of single cells as well as the evolution of species. Some recent developments in instrumentation, methodology, and applications of the PCR are presented in this review.

Binding Sites↗