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Clinical and Diagnostic Laboratory... Jul 2001To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes...
To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), from microglial cells was observed. Trophozoites of pathogenic A. culbertsoni made contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-alpha secreted from microglial cells cocultured with A. culbertsoni trophozoites or lysate increased at 6 h of incubation. The amounts of IL-1beta secreted from microglial cells cocultured with A. culbertsoni trophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation with A. culbertsoni lysate. These results suggest that pathogenic A. culbertsoni induces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-alpha and IL-1beta from microglial cells.
Topics: Acanthamoeba; Animals; Cell Membrane; Cell Nucleus; Cells, Cultured; Interleukin-1; Microglia; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha
PubMed: 11427438
DOI: 10.1128/CDLI.8.4.837-840.2001 -
Journal of Clinical Microbiology Oct 2015Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential for limiting ocular morbidity. We undertook a quality improvement... (Comparative Study)
Comparative Study
Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential for limiting ocular morbidity. We undertook a quality improvement initiative with respect to the molecular detection of acanthamoebae in our laboratory because of an unusual case of discordance. Nine ATCC strains of Acanthamoeba and 40 delinked, biobanked, surplus corneal scraping specimens were analyzed for the presence of acanthamoebae with four separate real-time PCR assays. The assay used by the Free-Living and Intestinal Amebas Laboratory of the CDC was considered the reference standard, and the performance characteristics of each individual assay and pairs of assays were calculated. Outcome measures were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of 49 included specimens, 14 (28.6%) were positive by the gold standard assay, and 35 (71.4%) were negative. The sensitivities of the individual assays ranged from 64.3% to 92.9%, compared to the gold standard, while the specificities ranged from 88.6% to 91.4%. The PPVs and NPVs ranged from 69.2% to 78.6% and from 86.1% to 96.9%, respectively. Combinations of assay pairs led to improved performance, with sensitivities ranging from 92.9% to 100% and specificities ranging from 97.1% to 100%. ATCC and clinical strains of Acanthamoeba that failed to be detected by certain individual assays included Acanthamoeba castellanii, Acanthamoeba culbertsoni, and Acanthamoeba lenticulata. For three clinical specimens, false negativity of the gold standard assay could not be excluded. Molecular diagnostic approaches, especially combinations of highly sensitive and specific assays, offer a reasonably performing, operator-independent, rapid strategy for the detection of acanthamoebae in clinical specimens and are likely to be more practical than either culture or direct microscopic detection.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Algorithms; Humans; Molecular Diagnostic Techniques; Predictive Value of Tests; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Time Factors
PubMed: 26202123
DOI: 10.1128/JCM.01607-15 -
The Journal of Protozoology 1992Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to...
Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti-Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba.
Topics: Acanthamoeba; Acquired Immunodeficiency Syndrome; Adult; Amebiasis; Animals; Antigens, Protozoan; Brain; Child; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunoblotting; Isoenzymes; Male; Peptides; Protozoan Proteins
PubMed: 1522539
DOI: 10.1111/j.1550-7408.1992.tb04853.x -
Molecules and Cells Oct 1999Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more... (Comparative Study)
Comparative Study
Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more active in pathogenic strains of amoeba whereas serine proteinases are found in both pathogenic and nonpathogenic strains. Cysteine proteinases thus play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culbertsoni by performing reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 1359 bp. The deduced amino acid sequence has the sequence homology with the cysteine proteinase genes of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathepsin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respectively. Sequence analysis and alignment showed significant similarity to other eukaryotic cysteine proteinases, including the conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. A 1.5 kbp mRNA was detected on Northern blot analysis using full-length cysteine proteinase cDNA as a probe. The A. culbertsoni cysteine proteinase gene (AcCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/threonine-rich C-terminal extension. Therefore, it has cathepsin L-like characteristics. Phylogenetic analysis based on the amino acid sequences of cysteine proteinase indicated that AcCP2 was closely related with papaya, while it was remotely related with those of Schistosoma.
Topics: Acanthamoeba; Amino Acid Sequence; Animals; Base Sequence; Biological Evolution; Blotting, Northern; Cathepsin L; Cathepsins; Cloning, Molecular; Cysteine Endopeptidases; DNA, Complementary; Endopeptidases; Genes, Protozoan; Helminth Proteins; Humans; Molecular Sequence Data; Open Reading Frames; Phylogeny; Plant Proteins; Protozoan Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity
PubMed: 10597037
DOI: No ID Found -
The Korean Journal of Parasitology Apr 2022Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary...
Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Antibodies; Humans; Peptides; Periplasmic Binding Proteins; Staphylococcus aureus; Trophozoites
PubMed: 35500897
DOI: 10.3347/kjp.2022.60.2.143 -
Pathogens (Basel, Switzerland) Jun 2020Amoebae of the genus are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, trophozoites were immunolocalized in...
Amoebae of the genus are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells-Schwann cells (SC). In this study, we analyzed in greater detail the in vitro early morphological events (1, 2, 3, and 4 h) during the interaction of trophozoites (ATCC 30171) with SC from (ATCC CRL-2941). Samples were processed for scanning and transmission electron microscopy as well as confocal microscopy. After 1 h of interaction, amoebae were observed to be adhered to the SC cultures, emitting sucker-like structures associated with micro-phagocytic channels. In addition, evidence of necrosis was identified since edematous organelles as well as multivesicular and multilamellar bodies characteristics of autophagy were detected. At 2 h, trophozoites migrated beneath the SC culture in which necrosis and autophagy persisted. By 3 and 4 h, extensive lytic zones were observed. SC necrosis was confirmed by confocal microscopy. We reported for the first time the induction of autophagic and necrotic processes in PNS cells, associated in part with the contact-dependent pathogenic mechanisms of trophozoites.
PubMed: 32526974
DOI: 10.3390/pathogens9060458 -
Clinical and Diagnostic Laboratory... Jul 2001Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological...
Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological comparisons. Previous serological studies have utilized a variety of antigen preparations and assay methods and reported disparate (3 to 100%) results. This study was designed to (i) optimize an enzyme-linked immunosorbent assay for detecting serum antibodies to each of the Acanthamoeba serogroups and (ii) test 55 healthy individuals for specific immunoglobulin G reactivity. The highest signal-to-background ratio was found when 3,000 fixed, intact trophozoites per well were used with a 1:10 serum dilution. Sera yielding optical densities of <0.25 against all three Acanthamoeba serogroups were used to define the cutoff for positive results. The highest background reactivity with these sera was seen with Acanthamoeba polyphaga (serogroup 2), followed by Acanthamoeba culbertsoni (serogroup 3) and Acanthamoeba astronyxis (serogroup 1). Of 55 subjects tested, the highest number of positive results was seen with A. polyphaga (81.8%), followed by A. astronyxis (52.8%) and A. culbertsoni (40%). Seven serum samples (12.7%) were negative for all three Acanthamoeba serogroups, 16 (29.1%) were positive for one serogroup only, 16 were positive for two serogroups, and 16 reacted to all three serogroups. Further analysis showed no significant associations between serogroup reactivity and age or gender. However, some ethnic differences were noted, especially with A. polyphaga antigens. In that case, serum samples from Hispanic subjects were 14.5 times less likely to be positive (P = 0.0025) and had lower mean absorbance values (P = 0.047) than those from Caucasian subjects. Overall, these data suggest that Acanthamoeba colonization or infection is more common than previously thought. Mild or asymptomatic infections may contribute to the observed serum reactivities.
Topics: Acanthamoeba; Adult; Amebiasis; Animals; Antibodies, Protozoan; Antigens, Protozoan; Enzyme-Linked Immunosorbent Assay; Female; Health Status; Humans; Male; Middle Aged; Rabbits; Reproducibility of Results
PubMed: 11427418
DOI: 10.1128/CDLI.8.4.724-730.2001 -
Infection and Immunity Jan 1975Antigens prepared from each of five strains (CA, CJ, HB-1, HB-3, and TY) of pathogenic Naegleria and the EG strain of nonpathogenic Naegleria gruberi were compared by... (Comparative Study)
Comparative Study
Antigens prepared from each of five strains (CA, CJ, HB-1, HB-3, and TY) of pathogenic Naegleria and the EG strain of nonpathogenic Naegleria gruberi were compared by the gel diffusion and immunoelectrophoresis techniques. Axenically grown amoebae were used as sources of antigens. Antisera were produced in individual rabbits against three strains (CA, CJ, and HB-1) of pathogenic Naegleria and the EG strain of N. gruberi. In the gel diffusion experiment each of the six antigens was reacted with each of the four antisera in agar gel. The results of these experiments revealed that the antigens of N. gruberi reacted strongly with the homologous antiserum but minimally with each of the three heterologous antisera. The antigens of all five pathogenic strains reacted extensively with the anti-CA, anti-CJ, and anti-HB-1 sera and moderately with the anti-EG serum. In the immunoelectrophoresis test each of the six antigens was separated electrophoretically in agar gel and reacted with each of the four antisera. The EG strain reacted extensively with its homologous antiserum and produced multiple precipitin arcs; it reacted minimally with anti-CA, anti-CJ, and anti-HB-1 sera and produced only three arcs. The antigens of all five strains of Naegleria fowleri reacted very strongly with anti-CA, anti-CJ, and anti-HB-1 sera and produced multiple precipitin arcs. They, however, reacted variably with the anti-EG serum and produced three to six precipitin arcs. Comparative immunoelectrophoretic analysis carried out on the CA and HB-1 strains revealed the antigenic identity of these two strains. Based on these results, together with those from the reciprocal absorption experiments, it was concluded that (i) the pathogenic strains of Naegleria, though they shared three to six common antigens with N. gruberi, were nevertheless distinct from it, and (ii) the five pathogenic strains were antigenically close and belonged in the same species. Antigens of Acanthamoeba castellanii, A. culbertsoni, and Entamoeba histolytica were also reacted with the four anti-Naegleria sera in gel diffusion experiments. Results of these tests indicate that these three organisms are antigenically distinct from Naegleria.
Topics: Absorption; Amoeba; Animals; Antigens; Entamoeba; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Rabbits
PubMed: 803926
DOI: 10.1128/iai.11.1.95-108.1975 -
Clinical and Experimental Immunology Nov 1983Normal human serum (NHS) contained an amoebicidal property for Acanthamoeba culbertsoni. Killing was quantitated by measuring the ability of the amoebae to undergo cell...
Normal human serum (NHS) contained an amoebicidal property for Acanthamoeba culbertsoni. Killing was quantitated by measuring the ability of the amoebae to undergo cell division subsequent to exposure to NHS, and also by microscopical examination. Plasma membrane disruption and extrusion of intracellular components occurred within 5-10 min following exposure to NHS. Adsorption of specific antibody did not remove the amoebicidal activity while heating serum at 56 degrees C/30 min or treatment with zymosan prevented the killing of A. culbertsoni. Haemolytic complement was consumed and C3 conversion occurred during the incubation of NHS with amoebae. Killing required the presence of the late complement components. The findings that (a) amoebae were killed in C2 deficient human serum and ethylene glycol tetra-acetic acid (EGTA), but not ethylenediamine tetra-acetic acid (EDTA) treated NHS; (b) haemolytic complement consumption, which occurred by incubating NHS with the amoebae, could be prevented by addition of EDTA, but not EGTA and (c) conversion of C3 occurred in the presence of EGTA, but not EDTA, indicated that activation of the alternative pathway of complement was involved. This may be of importance as a natural defence mechanism in humans against A. culbertsoni infections.
Topics: Amoeba; Animals; Blood Physiological Phenomena; Complement Activation; Complement C3; Complement Fixation Tests; Complement Pathway, Alternative; Edetic Acid; Egtazic Acid; Hot Temperature; Humans; Immunoelectrophoresis, Two-Dimensional; Microscopy, Electron; Zymosan
PubMed: 6418422
DOI: No ID Found -
Infection and Immunity Dec 1988The data showed that pathogenic free-living amoebae contain the proteolytic enzyme elastase. The levels of enzyme were similar in Naegleria fowleri, N. australianis...
The data showed that pathogenic free-living amoebae contain the proteolytic enzyme elastase. The levels of enzyme were similar in Naegleria fowleri, N. australianis italica, and Acanthamoeba culbertsoni A-1. No difference was found between elastase levels in a highly pathogenic N. fowleri and those in the same organism which had lost pathogenicity as a result of long-term axenic maintenance.
Topics: Acanthamoeba; Animals; Naegleria; Pancreatic Elastase
PubMed: 3182083
DOI: 10.1128/iai.56.12.3320-3321.1988