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PubMed2005

A metalloproteinase of Coccidioides posadasii contributes to evasion of host detection.

Coccidioides posadasii is a fungal respiratory pathogen of humans that can cause disease in immunocompetent individuals. Coccidioidomycosis ranges from a mild to a severe infection. It is frequently characterized either as a persistent disease that requires months to resolve or as an essentially asymptomatic infection that can reactivate several years after the original insult. In this report we describe a mechanism by which the pathogen evades host detection during the pivotal reproductive (endosporulation) phase of the parasitic cycle. A metalloproteinase (Mep1) secreted during endospore differentiation digests an immunodominant cell surface antigen (SOWgp) and prevents host recognition of endospores during the phase of development when these fungal cells are most vulnerable to phagocytic cell defenses. C57BL/6 mice were immunized with recombinant SOWgp and then challenged with a mutant strain of C. posadasii in which the MEP1 gene was disrupted. The animals showed a significant increase in percent survival compared to SOWgp-immune mice challenged with the parental strain. To explain these results, we proposed that retention of SOWgp on the surfaces of endospores of the mutant strain in the presence of high titers of antibody to the immunodominant antigen contributes to opsonization, increased phagocytosis, and killing of the fungal cells. In vitro studies of the interaction between a murine alveolar macrophage cell line and parasitic cells coated with SOWgp showed that the addition of anti-SOWgp antibody could enhance phagocytosis and killing of Coccidioides. We suggest that Mep1 plays a pivotal role as a pathogenicity determinant during coccidioidal infections and contributes to the ability of the pathogen to persist within the mammalian host.

Animals↗

PubMed1996

Immunoelectrochemical assays for bacteria: use of epifluorescence microscopy and rapid-scan electrochemical techniques in development of an assay for Salmonella.

Immunoelectrochemical sensors in which the sensor surface functions as both analyte capture phase and electrochemical detector have recently been developed for bacteria analysis. The speed and sensitivity of these devices make them very attractive for applications such as the detection of pathogenic microorganisms in food and water. However, the development and optimization of assays utilizing these sensors can be complicated by undesired interactions between the capture and detection functions. Modification of the sensor to achieve improvements in one function can have deleterious effects on the other function, and such effects can be difficult to diagnose and correct. In the course of investigations on immunoelectrochemical detection of Salmonella, we developed a rapid, nondestructive epifluorescence microscopy method to determine bacteria capture efficiency. This method enabled us to study capture and detection functions independently and efficiently identify performance-limiting factors. Rapid-scan electrochemical methods were used to optimize detection sensitivity and to provide diagnostic information on detection performance.

Electrochemistry↗

PubMed2004

The role and regulation of programmed cell death in plant-pathogen interactions.

It is commonly known that animal pathogens often target and suppress programmed cell death (pcd) pathway components to manipulate their hosts. In contrast, plant pathogens often trigger pcd. In cases in which plant pcd accompanies disease resistance, an event called the hypersensitive response, the plant surveillance system has learned to detect pathogen-secreted molecules in order to mount a defence response. In plants without genetic disease resistance, these secreted molecules serve as virulence factors that act through largely unknown mechanisms. Recent studies suggest that plant bacterial pathogens also secrete antiapoptotic proteins to promote their virulence. In contrast, a number of fungal pathogens secrete pcd-promoting molecules that are critical virulence factors. Here, we review recent progress in determining the role and regulation of plant pcd responses that accompany both resistance and susceptible interactions. We also review progress in discerning the mechanisms by which plant pcd occurs during these different interactions.

Apoptosis↗

PubMedDate not supplied

[Value of microbiologic studies for diagnosis of post-enteritis reactive arthritis].

Reactive arthritis may develop within a period of some days until upto 3 weeks after infections with Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni/coli, Shigella and Salmonella. Intestinal infections with Klebsiella pneumoniae, Clostridium perfringens, Clostridium difficile, Cryptosporidium, Strongyloides stercoralis, Taenia saginata and Schistosoma mansoni are, in some cases, considered to be responsible for reactive arthritis. Detection of pathogenic bacteria in feces is generally most successful in the early stage of the infection. A large spectrum of special tests is required in order to detect all the causative agents and to ensure reliable results. It is therefore necessary that the laboratory is provided with information about the diagnostic object and the tentative diagnosis. The detection of serum antibodies to Y. enterocolitica, Y. pseudotuberculosis, C. jejuni/coli, and Schistosoma may suggest connections between infection and reactive arthritis.

Arthritis, Infectious↗

PubMed1998

Microbiota associated with experimental peri-implantitis and periodontitis in adult Macaca mulatta monkeys.

This study examines the microbiota associated with the progression of experimental peri-implantitis and periodontitis induced concurrently in partially edentulous adult monkeys. Root-form and plate-form implants with fixed prosthesis in place for at least 12 months and their corresponding opposite molar teeth were ligated for 6 months. The microbiota in plaque around these ligated dental implants and molars were studied at 0, 1, 2, 3, and 6 months post-ligation. Plaque samples were analyzed by dark-field microscopy and selective and non-selective culture. Putative periodontal pathogens were detected as a major component of the microbiota cultured from plaque samples obtained from experimental peri-implantitis sites. Overall, the types and relative proportions of putative periodontal pathogens in plaque associated with ligature-induced peri-implantitis and ligature-induced periodontitis were similar. Only levels of anaerobic Actinomyces and spirochetes were significantly different between both sites. Spirochete levels were significantly higher at peri-implantitis sites when compared with levels at periodontitis sites after 6 months, and spirochete levels increased significantly between 0 and 6 months post-ligation at implant sites. Levels of spirochetes correlated significantly with probing depth and bone loss at peri-implantitis sites. Overall, Actinomyces levels were higher at periodontitis sites. Porphyromonas species were not detected continuously as part of the peri-implantitis microbiota. In conclusion, this study finds that the microbiota associated with the progression of experimental peri-implantitis and periodontitis occurring concurrently in partially edentulous mouths are similar.

Actinomyces↗

PubMed1998

Lack of utility of the lysis-centrifugation blood culture method for detection of fungemia in immunocompromised cancer patients.

We retrospectively compared the utility of a fungal isolation device (Isolator) versus conventional techniques for recovering fungal organisms from blood cultures obtained from neutropenic cancer patients. Positive cultures were deemed true pathogens, possible pathogens, or contaminants according to laboratory and clinical criteria. Fifty-three patients had 66 positive blood cultures for fungi, nine on multiple occasions. In 20 episodes true pathogens were recovered, 6 from broth medium alone, 4 from the Isolator system alone, and 10 from both systems. False-negative cultures were noted in 4 of 20 (20%) cases in which broth medium was used and in 6 of 20 (30%) cases in which the Isolator system was used. Possible pathogens were detected in 4 of 66 blood culture-positive cases. Forty-two positive cultures were considered contaminants, 1 collected from standard medium and 41 of 42 (98%) which grew only in Isolators. Eleven of 18 patients with true fungal infections expired as a result of infection, while 4 of 33 patients with a contaminant expired, none from a fungal cause. We do not advocate the routine use of Isolator tubes in the evaluation of the febrile, neutropenic patient due to the high rates of false positives and of contamination.

Adult↗

PubMed1997

Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay.

A recombinant enzyme-linked immunosorbent assay (ELISA) system, entirely based on antibody fragments, is described here as an attractive alternative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single-chain Fv (scFv) fusion proteins suitable as coating and detecting reagents, respectively, in a highly sensitive double antibody sandwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization. The detection reagent is a fusion protein, in which a modified Escherichia coli alkaline phosphatase with increased specific activity is attached to the carboxy-terminus of the scFv. This conjugate is produced using pDAP2/S as cloning and expression vector. Optimized bacteria expression and simple one-step purification by hexahistidine tag-mediated metal chelate affinity chromatography yielded milligrams of ELISA reagent per liter of bacterial culture in both cases. A double antibody sandwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion proteins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was detected with a sensitivity higher than that reached in a conventional ELISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series, allowing simple subcloning after isolation of scFv from phage display libraries. It is therefore easy to develop and produce an ELISA entirely by using bacterial recombination and expression techniques.

Alkaline Phosphatase↗

PubMed1984

An enveloped virus in stools of children and adults with gastroenteritis that resembles the Breda virus of calves.

Pleomorphic virus-like particles about 100 nm in diameter with a fringe of closely applied peplomers (7-9 nm in length) were observed by electron microscopy in the stools of 20 children and adults with gastroenteritis. In most of the samples no other viral or bacterial pathogens were detected. In form and under immune electron microscopy these virus-like particles resembled the Breda virus isolated from diarrhoeic calves. These objects may be a viral pathogen of humans.

Adult↗

PubMed2006

Does selection by resistant hosts trigger local adaptation in plant-pathogen systems?

Understanding the consequences of selection by host resistance on pathogen population structure provides useful insights into the dynamics of host-parasite co-evolution processes and is crucial for effective disease management through resistant cultivars. We tested general vs. local population adaptation to host cultivars, by characterizing a French collection of Phytophthora infestans (the causal organism of potato late blight) sampled during two consecutive years on cultivars exhibiting various levels of resistance. Local populations were structured by the host for virulence (qualitative pathogenicity) but also for aggressiveness (quantitative pathogenicity). All populations had a low genotypic diversity for amplified fragment length polymorphisms (AFLPs), and presumably consisted of a few closely related clonal lineages. No correlation was detected between pathogenicity traits and AFLP genotypes. The data support the hypothesis of general adaptation for aggressiveness, to which directional selection for virulence is superimposed when race-specific resistance is introduced.

DNA Fingerprinting↗

PubMed1986

Cryptosporidiosis among black children in hospital in South Africa.

Over a period of 3 months during the summer, 362 African children admitted to King Edward VIII Hospital, Durban, were screened for the faecal excretion of Cryptosporidium oocysts. Of 259 children with diarrhoea, oocysts were detected in 31 (11.9%), while none was found in the faeces of 103 children without diarrhoea (controls). All those children excreting Cryptosporidium were under 2 years of age, giving a prevalence of 15% for this group. Other potential enteric pathogens were detected in the faeces of 12 (38.7%) of these children. The case fatality rate for patients with Cryptosporidium was 22.6%, which may reflect the selection of patients in a study concentrating on hospital inpatients. Cryptosporidium was the second most common organism detected in diarrhoeal faeces, and the only one detected in 9.2% of diarrhoeal children aged less than two years. These findings indicate that Cryptosporidium should be regarded as a potential pathogen in children admitted to this hospital with severe diarrhoea. Such association of Cryptosporidium with diarrhoea in children accords with recent studies in other parts of the world.

Age Factors↗

PubMed2002

Fish chromatophores as cytosensors in a microscale device: detection of environmental toxins and bacterial pathogens.

Fish chromatophores from Betta splendens are used as the cytosensor element in the development of a portable microscale device capable of detecting certain environmental toxins and bacterial pathogens by monitoring changes in pigment granule distribution. The adaptation of chromatophores to a microscale environment has required the development of enabling technologies to produce miniaturized culture chambers, to integrate microfluidics for sample delivery, to miniaturize image capture, and to design new statistical methods for image analyses. Betta splendens chromatophores were selected as the cytosensor element because of their moderate size, their toleration of close contact, and most importantly, for their responses to a broad range of chemicals and pathogenic bacteria. A miniaturized culture chamber has been designed that supports chromatophore viability for as long as 3 months, and that can be easily transported without damage to the cells. New statistical methods for image analyses have been developed that increase sensitivity and also decrease the time required for detection of significant changes in pigment granule distribution. Betta chromatophores have been tested for their responses to selected pathogenic bacteria and chemical agents. We discuss in detail the aggregation of pigment granules seen when chromatophores are incubated with Bacillus cereus, a common cause of food poisoning. Also described are the more subtle responses of chromatophores to a class of environmental chemical toxins, polynuclear aromatic hydrocarbons. We show that the chromatophores are able to detect the presence of certain polynuclear aromatic hydrocarbons at concentrations lower than the Environment Protection Agency (EPA) 550.1 standards.

Animals↗

PubMed2001

Identification of human enterovirulent Escherichia coli strains by multiplex PCR.

Some strains of Escherichia coli are involved in enteric infections in both adults and children. However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E. coli, because of the lack of phenotypic differences. In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E. coli pathotypes. Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions. This method was applied to the detection of pathogenic E. coli isolated from 90 patients' stools specimens during an 18-month survey. Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E. coli strain was detected. This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens.

Adolescent↗

PubMed1976

Erwinia salicis: its metabolism and variability in vitro, and a method to demonstrate the pathogen in the host.

Morphological, biochemical and serological features of eleven Erwinia salicis isolates were examined. Three groups were demonstrated, one of which comprises the Dutch isolates. Serological techniques proved to be a valuable addition to conventional plating techniques for detecting the pathogen. Willow wood extract with 5% sucrose, 0.06% "Lab Lemco" broth and 1.5% agar appeared to be a suitable medium for the isolation of E. salicis, because of its selectivity.

Culture Media↗

PubMed1999

Isolation of respiratory bacterial pathogens from the throats of healthy infants fed by different methods.

Most bacterial infections are caused by organisms that have already colonized the host. Bacterial attachment to pharyngeal cells and proliferation may be necessary to infect the lower respiratory tract or middle ear. We investigated the incidence of pathogenic bacteria isolated from the throat of healthy infants with different feeding methods. The protecting role of breastmilk is also discussed. The incidence of respiratory bacterial pathogens isolated from the oropharynx of 113 normal infants with different feeding methods was investigated. Group A beta haemolytic Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were selected as respiratory bacterial pathogens. No respiratory bacterial pathogens were detected in breastfed and mixed-fed infants. Haemophilus influenzae and Moraxella catarrhalis were isolated from the oropharynx of formula-fed infants. The incidence of respiratory bacterial pathogens did differ among infants with different feeding methods. These results suggest that breastmilk may inhibit the colonization by respiratory bacterial pathogens of the throat of infants, by enhancing mucosal immunity against respiratory tract infection.

Bacteria↗

PubMed2005

Bacteria and inflammatory cells in fetal membranes do not always cause preterm labor.

Intrauterine infection has been frequently linked with preterm labor before 30 wk of human pregnancy. Many different species of organisms have been detected, leading to the suggestion that infection-induced preterm labor is a generic inflammatory response to organisms rather than a specific response to a limited number of pathogens. The detection of organisms by microbiological culture is a laborious and unreliable process, so the aim of this study was to harness modern molecular techniques to detect organisms in tissues from human pregnancy. A DNA probe specific for conserved regions of bacterial 16S ribosomal RNA sequence was designed and labeled with fluorescein for fluorescence in situ hybridization. Organisms were detected in the great majority (>80%) of fetal membranes after prolonged premature rupture of the fetal membranes and after preterm labor, which was consistent with previous data. Organisms were also detected in fetal membranes after preterm delivery without labor and in term deliveries (with or without labour). Inflammatory cells were found frequently in the amnion or chorion of preterm fetal membranes but not in term tissues. Our primary finding is that fluorescence in situ hybridization is an appropriate method to detect organisms in human fetal membranes. In addition, our data show that bacteria may be present in fetal membranes without necessarily causing an inflammatory response, so the mere presence of bacteria may not be sufficient to cause preterm labor.

Antigens, CD↗

PubMedDate not supplied

Body weight suppression in broilers naturally infected with avian leukosis virus subgroup J.

Broiler progeny were hatched from avian leukosis virus (ALV)-negative and ALV subgroup J (ALV-J)-positive breeders. ALV-J infection in progeny was identified by p27 antigen capture enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction specific for ALV-J performed on serum or plasma samples obtained at hatch. Hatch weights did not differ between ALV-negative broilers and ALV-J-positive broilers. Body weights of ALV-J-positive broilers were 64.4% of those of ALV-negative broilers at 1 wk of age. At 8 wk of age, weights of broilers with congenital ALV-J infection were still only 63.8% of those of ALV-negative broilers. No other concurrent pathogen was detected in either group of broilers. These findings indicate that congenital ALV-J infection is associated with significant weight suppression in broilers in the absence of other pathogens.

Animals↗

PubMed2002

Detection of the high-pathogenicity island of Yersinia enterocolitica in enterotoxigenic and enteropathogenic E.coli strains.

OBJECTIVE: To describe the distribution of high-pathogenicity island (HPI) of Yersinia enterocolitica in enterotoxingenic E.coli (ETEC) and enteropathogenic E.coli (EPEC), and to understand the structure and function of HPI. METHODS: PCR was used to detect irp2, fyua and asn-intB genes with subsequent sequence analysis of these genes. Nucleic acid in situ hybridization was employed to identify the specificity of irp2 and fyua. RESULTS: Thirty irp2-positive strains were isolated from 93 ETEC strains and 3 from 10 EPEC strains, making a positivity rate of 32.25% and 30% respectively, and the positivity rates of fyua gene in ETEC and EPEC were 21.51% and 30% respectively. In most of these positive isolates, HPI was bordered by an asn tRNA locus, as in Yersinia sp. CONCLUSIONS: This study demonstrates that the high positivity rate of HPI of Yersinia enterocolitica in ETEC and EPEC strains may be crucial to the virulence changes, virulence evolution and virulence regulation in E.coli.

Bacterial Outer Membrane Proteins↗

PubMed1991

Cryptosporidium in Bedouin and Jewish infants and children in southern Israel.

We conducted a 1-year prospective study in two clinics and a hospital in the Negev region of southern Israel to determine the epidemiological and clinical patterns of Cryptosporidium diarrhea among Jewish and Bedouin infants and children living in the same geographical area. A total of 612 episodes were studied: 398 in Bedouins and 214 in Jews, of which 449 occurred in patients with diarrhea and 164 in controls. Cryptosporidium was detected in 13 of 382 patients (3.4%) with diarrhea and in 1 of 138 controls (0.7%) (P = 0.078). In 5 of 13 Cryptosporidium-positive patients (38%) another pathogen was detected. No significant difference in Cryptosporidium detection rates was observed between Jews and Bedouins or between hospitalized or nonhospitalized patients. The frequency of Cryptosporidium detection did not differ significantly when three age-groups were compared (less than 6 months old, 7-12 and 13-36 months old). The rate of Cryptosporidium detection was similar among malnourished and well-nourished patients, as determined by weight-for-height percentiles. Cryptosporidium was detected more frequently during the summer months (8.3%) than during the rest of the year (1.2%) (P less than 0.001). Patients with Cryptosporidium diarrhea did not differ clinically from patients with other causes of diarrhea. However, they were characterized by the absence of fecal leukocytes. Cryptosporidium is not a rare cause of diarrhea in southern Israel. It is more prevalent during the hot and dry season and can be detected in a relatively high prevalence among very young infants. Its clinical features are indistinguishable from those of patients with non-cryptosporidial diarrhea.

Child, Preschool↗