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PubMedDate not supplied

Prevalence of enteropathogenic bacteria in surgically treated chronic inflammatory bowel disease.

BACKGROUND/AIMS: In order to assess the potential impact of bacterial eradication on recurrence rates, the prevalence of various enteropathogenic bacteria and toxins in chronic inflammatory bowel diseases (CIBD) was prospectively examined. METHODOLOGY: Stool, sera and gut tissue samples from a total of 59 patients (33 males, 26 females; mean age: 42 years +/- 14; 21 Crohn's disease, 14 ulcerative colitis, 24 controls) were examined for the presence of enteropathogenic bacteria by culture, immunoblotting and PCR. RESULTS: Conventional cultures failed to detect obligate pathogenic bacteria. By PCR, mycobacteria were found in 85% of all groups, with mycobacterium paratuberculosis not detected. Yersinia species were observed in 63% of patients with Crohn's disease, in 46% of patients with ulcerative colitis, and in 36% of the control patients. Pathogenic E. coli were identified in stool samples of three patients with ulcerative colitis (21%) by amplifying the EAE-gene, one of whom exhibited shiga-like-toxin as well. CONCLUSIONS: We concluded that mycobacteria do not play a causative role in CIBD. Yersinia species seem to persist in intestinal tissue in CIBD patients without adequate immune response and might, thus, contribute to tissue destruction. E. coli infections contribute to the disease process in a small group of patients with ulcerative colitis and their eradication might eliminate the need for immediate surgical intervention.

Adult↗
PubMed2006

Fitness consequences of infection of Arabidopsis thaliana with its natural bacterial pathogen Pseudomonas viridiflava.

Variation in plant resistance to pathogen infection is commonly observed in interactions between wild plants and their foliar pathogens. Models of host-pathogen interactions indicate that a large cost of infection is generally necessary to maintain this variation, yet there is limited evidence that foliar pathogens cause detectable fitness reductions in wild host plants. Most published work has focused on fungal pathogens. Pseudomonas viridiflava, a common bacterial pathogen of the annual weed Arabidopsis thaliana across its range, comprises two distinct genetic clades that cause disease symptoms of different severity. Here we measured the extent of infection of wild A. thaliana populations in the Midwest, USA, and examined the effect on seed production, in field and growth-chamber experiments, of experimental inoculation with isolates from the two clades. We found infection with P. viridiflava varied from 0 to 56% in Midwest A. thaliana populations, with the possibility of several leaves per plant infected later in the growing season. In the growth chambers, experimental inoculation reduced seed set by averages of 15 and 11% for clades A and B, respectively. In the field experiment, only clade A affected plant fitness significantly, reducing seed set by an average of 38%. Underlying these average effects we observed both negative and positive effects of infection, and variation in both fitness among plant genotypes and sensitivity to environmental conditions.

Arabidopsis↗
PubMedDate not supplied

First report of Escherichia coli O157 among Iraqi children.

We determined the prevalence of enterohaemorrhagic Escherichia coli, especially E. coli O157, and other enteropathogens among 200 children with bloody diarrhoea and 100 age-matched controls at two Baghdad hospitals. Bacterial and parasitic agents were found in 39.5% and 28.5% of cases, respectively; no pathogen was detected in 32%. E. coli O157 was identified in 11.5% and more than one pathogen was found in 15.5% of cases. The most common pathogens were enteropathogenic E. coli (EPEC) (5%); E. coli other than E. coli O157 or EPEC (15%); Entamoeba histolytica (25%) and Giardia lamblia (3.5%). All isolates of E. coli O157:H7 were sensitive to cephalexin, ciprofloxacin, gentamicin and nalidixic acid and resistant to erythromycin, polymyxin B and vancomycin. Resistance to 6 or more antimicrobial agents was common (50% of isolates).

Abdominal Pain↗
PubMed1998

In situ localisation of Yersinia enterocolitica by catalysed reported deposition signal amplification.

AIM: The sensitive detection of pathogenic Yersinia enterocolitica in paraffin embedded tissue sections by in situ hybridisation (ISH). METHODS: Y enterocolitica infected cell lines, rat spleens, and patient biopsy specimens were used to compare conventional ISH, immune fluorescence assay (IFA) detection, and catalysed reporter deposition (CARD) signal amplification ISH. RESULTS: CARD-ISH was shown to be more sensitive then conventional ISH and had a comparable sensitivity to IFA. In contrast to IFA, CARD-ISH preserved good tissue morphology. CONCLUSIONS: CARD-ISH appeared to be a fast and sensitive ISH method for detecting Y enterocolitica in routinely processed tissue sections. Application of this method allows the combination of routine detection and cellular localisation of the pathogen within the infected tissue.

Animals↗
PubMed2001

Infectious agents in coronary lesions obtained by endatherectomy: pattern of distribution, coinfection, and clinical findings.

BACKGROUND: Cytomegalovirus (CMV), Chlamydia pneumoniae (C. pneumoniae), and Helicobacter pylori (H. pylori) have been implicated in atherosclerosis and restenosis after angioplasty. The patterns of distribution within coronary lesions and possible coinfections of these pathogens in the coronary vasculature had not previously been evaluated. DESIGN: A prospective, observational clinical study. METHODS: Large coronary specimens (9-105 mm long) were obtained by endatherectomy of 53 patients undergoing aortocoronary bypass surgery. Samples were taken from two different sites of every lesion, resulting in a total of 106 probes. Presence of each pathogen was determined by polymerase chain reaction, subsequent hybridization, and DNA sequencing. RESULTS: Cytomegalovirus and C. pneumoniae were detected in 30 and 32% of the samples, respectively; H. pylori was not detectable. The pathogens were not homogeneously distributed. A concurrent infection with both pathogens was observed in five of 106 (5%) lesions and five of 53 (9%) patients. Restenotic lesions were more often found in specimens in which cytomegalovirus was detected (five of 16 versus two of 37). Patients with C. pneumoniae-positive coronary lesions more commonly presented with unstable angina. CONCLUSIONS: Inhomogeneous infections with cytomegalovirus and C. pneumoniae of coronary atherosclerotic lesions are found to be prevalent when serial analysis is performed. Concurrent infection with both pathogens occurs coincidentally; however, possible clinical implications of this new observation and the pathogenic impact on atherosclerosis need further investigation.

Aged↗
PubMed2002

Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR.

A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100 kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5' untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3 viruses/ml to 83 viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters.

Animals↗
PubMed1990

Superoxol and aminopeptidase tests for identification of pathogenic Neisseria species and Moraxella (Branhamella) catarrhalis.

The superoxol test, and prolyl aminopeptidase and gammaglutamyl aminopeptidase tests were evaluated for the detection of pathogenic Neisseria spp. using 317 strains of Neisseria-ceae. The superoxol test was positive for all 116 gonococci and 62 Moraxella (Branhamella) catarrhalis strains, but also for three strains of Neisseria meningitidis, one strain of Neisseria lactamica and eight saprophytic neisseriae. When using strains grown on Thayer-Martin medium, the positive and negative predictive values of the superoxol test for the identification of Neisseria gonorrhoeae were 96.7% and 100% respectively. Meningococci were the only neisseriae growing on Thayer-Martin medium that showed gamma-glutamyl aminopeptidase activity. The prolyl aminopeptidase test showed low specificity.

Aminopeptidases↗
PubMed2003

Role of Toll-like receptors in pathogen recognition.

The innate immune system relies on a vast array of non-clonally expressed pattern recognition receptors for the detection of pathogens. Pattern recognition receptors bind conserved molecular structures shared by large groups of pathogens, termed pathogen-associated molecular patterns. The Toll-like receptors (TLRs) are a recently discovered family of pattern recognition receptors which show homology with the Drosophila Toll protein and the human interleukin-1 receptor family. Engagement of different TLRs can induce overlapping yet distinct patterns of gene expression that contribute to an inflammatory response. The TLR family is characterized by the presence of leucine-rich repeats and a Toll/interleukin-1 receptor-like domain, which mediate ligand binding and interaction with intracellular signaling proteins, respectively. Most TLR ligands identified so far are conserved microbial products which signal the presence of an infection, but evidence for some endogenous ligands that might signal other danger conditions has also been obtained. Molecular mechanisms for pathogen-associated molecular pattern recognition still remain elusive but seem to be more complicated than initially anticipated. In most cases, direct binding of microbial ligands to TLRs still has to be demonstrated. Moreover, Drosophila TLRs bind endogenous ligands, generated through a proteolytic cascade in response to an infection. In the case of endotoxin, recognition involves a complex of TLR4 and a number of other proteins. Moreover, TLR heterodimerization further extends the spectrum of ligands and modulates the response towards specific ligands. The fact that TLR expression is regulated in both a cell type- and stimulus-dependent fashion further contributes to the complexity.

Animals↗
PubMed1999

A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.

Animals↗
PubMed1992

Transfer of both protection and delayed-type hypersensitivity against live Listeria is mediated by the CD8+ T cell subset: a study with Listeria-specific T lymphocytes recovered from murine infected liver.

The recruitment of specific T lymphocytes in murine liver is thought to be a key event in the ultimate control of Listeria monocytogenes growth during primary infection. However, there has been little functional characterization of the cell populations recruited in this non-lymphoid organ. Therefore in this study, the recruited lymphomyeloid cells were isolated from the liver of C57BL/6 mice at the peak of the immune response (day 7) triggered by a non-lethal L.monocytogenes infection. The anti-Listeria T lymphocytes were detected in vivo by their ability to transfer protection and delayed-type hypersensitivity (DTH) to live L.monocytogenes in naive recipients: protection was measured not only by the effect on reduction of the bacterial load in liver and spleen, but also on survival after the lethal challenge, and DTH was detected using as eliciting antigen, either live L.monocytogenes or heat-killed L.monocytogenes. When live pathogens were used, both functions were found to be mediated by T lymphocytes belonging to the CD8+ subset. However, when heat-killed L.monocytogenes were used as eliciting antigen in the DTH assay, Listeria-specific CD8+ T lymphocytes could not be restimulated in immune lymphoid cell populations recovered either from liver or spleen of Listeria-infected mice. Both populations were thus found to share the same qualitative properties in the DTH assay. The importance of the use of live pathogens versus heat-killed pathogens for detection of DTH and protection functions is discussed in the light of current concepts on processing and presentation pathways of Listeria-derived peptides.

Animals↗
PubMed1997

Towards the routine application of nucleic acid technology for avian disease diagnosis.

The use of nucleic acid technology (polymerase chain reaction, probing, restriction fragment analysis and nucleotide sequencing) in the study of avian diseases has largely been confined to fundamental analysis and retrospective studies. More recently these approaches have been applied to diagnosis and what one might call real-time epidemiological studies on chickens and turkeys. At the heart of these approaches is the identification and characterisation of pathogens based on their genetic material, RNA or DNA. Among the objectives has been the detection of pathogens quickly combined with the simultaneous identification of serotype, subtype or genotype. Nucleic acid sequencing also gives a degree of characterisation unmatched by other approaches. In this paper we describe the use of nucleic acid technology for the diagnosis and epidemiology of infectious bronchitis virus, turkey rhinotracheitis virus (avian pneumovirus) and Newcastle disease virus.

Animals↗
PubMed1992

Detection of bacteriuria and pyuria by URISCREEN a rapid enzymatic screening test.

A multicenter study was performed to evaluate the ability of the URISCREEN (Analytab Products, Plainview, N.Y.), a 2-min catalase tube test, to detect bacteriuria and pyuria. This test was compared with the Chemstrip LN (BioDynamics, Division of Boehringer Mannheim Diagnostics, Indianapolis, Ind.), a 2-min enzyme dipstick test; a semiquantitative plate culture method was used as the reference test for bacteriuria, and the Gram stain or a quantitative chamber count method was used as the reference test for pyuria. Each test was evaluated for its ability to detect probable pathogens at greater than or equal to 10(2) CFU/ml and/or greater than or equal to 1 leukocyte per oil immersion field, as determined by the Gram stain method, or greater than 10 leukocytes per microliter, as determined by the quantitative count method. A total of 1,500 urine specimens were included in this evaluation. There were 298 specimens with greater than or equal 10(2) CFU/ml and 451 specimens with pyuria. Of the 298 specimens with probable pathogens isolated at various colony counts, 219 specimens had colony counts of greater than or equal to 10(5) CFU/ml, 51 specimens had between 10(4) and 10(5) CFU/ml, and 28 specimens had between 10(2) and less than 10(4) CFU/ml. Both the URISCREEN and the Chemstrip LN detected 93% (204 of 219) of the specimens with probable pathogens at greater than or equal to 10(5) CFU/ml. For the specimens with probable pathogens at greater than or equal to 10(2) CFU/ml, the sensitivities of the URISCREEN and the Chemstrip LN were 86% (256 of 298) and 81% (241 of 298), respectively. Of the 451 specimens with pyuria, the URISCREEN detected 88% (398 of 451) and Chemstrip LN detected 78% (350 if 451). There were 204 specimens with both greater than or equal to 10(2) CFU/ml and pyuria; the sensitivities of both methods were 95% (193 of 204) for these specimens. Overall, there were 545 specimens with probable pathogens at greater than or equal to 10(2) CFU/ml and/or pyuria. The URISCREEN detected 85% (461 of 545), and the Chemstrip LN detected 73% (398 of 545). A majority (76%) of the false-negative results obtained with either method were for specimens without leukocytes in the urine. There were 955 specimens with no probable pathogens or leukocytes. Of these, 28% (270 of 955) were found positive by the URISCREEN and 13% (122 of 955) were found positive by the Chemstrip LN. A majority of the false-positive results were probably due, in part, to the detection of enzymes present in both bacterial and somatic cells by each of the test systems. Overall, the URISCREEN is rapid, manual, easy-to-perform enzymatic test that yields findings similar to those yielded by the Chemstrip LN for specimens with both greater than or equal to 10(2) CFU/ml and pyuria or for specimens with greater than or equal to 10(5) CFU/ml and with or without pyuria. However, when the data were analyzed for either probable pathogens at less 10(5) CFU/ml or pyuria, the sensitivity of the URISCREEN was higher (P less than 0.05).

Bacteriological Techniques↗
PubMed2003

An evaluation of molecular diagnostic tools for the detection and differentiation of human-pathogenic Cryptosporidium spp.

The performance of 10 commonly used genotyping tools in the detection and differentiation of 7 human-pathogenic Cryptosporidium spp. (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris and Cryptosporidium pig genotype 1) was evaluated. All 3 SU rRNA gene-based tools could amplify the DNA of 7 Cryptosporidium spp. efficiently. However, the tools based on the antigens TRAP-C1, TRAP-C2 and COWP genes, the housekeeping genes HSP70 and DHFR, or a genomic sequence, failed to detect the DNA of C. felis, C. canis, Cryptosporidium pig genotype I, and C. muris. With the exception of 1 tool based on the TRAP-C2 gene, the PCR-RFLP or the PCR sequencing tools evaluated in this study could differentiate C. hominis, C. parvum and C. meleagridis from each other, and 2 SSU rRNA gene-based tools could differentiate all 7 Cryptosporidium spp. Thus, a thorough understanding of the strength and weakness of each technique is needed when using molecular diagnostic tool in epidemiological investigations of human cryptosporidiosis.

Animals↗
PubMed2001

PCR detection of 5 putative periodontal pathogens in dental plaque samples from children 2 to 12 years of age.

BACKGROUND, AIMS: The purpose of this study was to detect the presence of Prevotella intermedia, P. nigrescens, Bacteroides forsythus, Treponema denticola, and Campylobacter rectus in plaque samples from 119 children, collected from their toothbrushes using a polymerase chain reaction (PCR). METHOD: The subjects were 24, 83, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from the plaque samples was approximately 640 microg/ml, which was deemed sufficient for performing a PCR-based survey. RESULTS: The prevalence by PCR in healthy, gingivitis, and periodontitis subjects was 0.0%, 6.0% and 25.0% for P. intermedia, 45.8%, 79.5% and 50.0% for P. nigrescens, 33.3%, 63.9% and 58.3% for B. forsythus, 0.0%, 18.1% and 16.7% for T. denticola, and 100% in duplicate for C. rectus, respectively. CONCLUSION: Our survey indicated that P. intermedia and T. denticola were more associated with periodontal diseases, B. forsythus and P. nigrescens had a moderate prevalence in all clinical groups, while C. rectus were the most commonly detected species in the oral cavities of children suggesting establishment in their early years.

Age Factors↗
PubMed2004

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses.

A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.

Child, Preschool↗
PubMed1995

Local succession of adenovirus strains in pediatric gastroenteritis.

Assessment of the current incidence of different adenovirus types in local gastroenteritis involved the examination of over 1,000 stool specimens annually from 1988-1992. Adenoviruses were detected by electron microscopy and/or cell culture in 32% of the specimens in which a viral pathogen was detected. The identification of every adenovirus isolate to type by neutralization with specific antisera against the first 6 types and by restriction analysis of nonneutralized isolates was started in 1990. Samples from 1988 and 1989 were examined retrospectively. Adenovirus strains were compared to those isolated in a study between 1980-1983. Enumeration of individual adenovirus types revealed a number of trends, demonstrating that rapid changes in the local incidence of several strains were occurring in Manitoba. The incidence of adenovirus type 40 (Ad40) as a cause of gastroenteritis was found to have fallen dramatically in recent years. The predominant cause of gastroenteritis in Manitoba is a variant strain of Ad41, increasing in predominance each year and now responsible for over a third of the symptomatic cases examined since 1990. The majority of restriction site differences of the Ad41 variant strain from the prototype strain Tak were mapped to the hexon and fiber genes, both of which code for the neutralizable external viral epitopes. The probability of the observed pattern of mutations occurring by chance was calculated as P < 0.0005, indicating a strong pressure for selection of these immunologically significant alterations to the viral proteins responsible for cell attachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus Infections, Human↗
PubMed1997

Detection of cross-reactive antigens shared by Fusarium oxysporum and Glycine max by indirect ELISA and their cellular location in root tissues.

Pathogenicity test of Fusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium of F. oxysporum. Polyclonal antisera were raised against the mycelial suspension of F. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.

Antigens↗