Aspects of the Biology of Small Free-Living and Facultative Parasitic Amoebae

Aspects of the biology of small, naked, free-living amoebae which include some facultatively parasitic forms have been studied with emphasis on two selected genera, Naegleria and Hartmannella. In the former genus, non-pathogenic Naegleria gruheri (CCAP strain 1518/1 A), as well as the closely related pathogenic Naegleria fowleri, have been investigated. A combination of fluorescence and electron microscopy techniques have been employed to investigate mitosis in N. gruberi with special reference to the distribution of chromosomes/DNA and the possible existence of the microtubule organising centres. In transmission electron micrographs, the nucleolus appeared to persist during nuclear divison but its electron density changed during the course of nuclear division. During most of stages of nuclear division, chromosomal elements could not be distinguished with certainty from nucleolar material. Possible chromosomal structures could be observed as electron dense spherical profiles or as fluorescent dots only when the nucleus was at metaphase or anaphase stages.Intranuclear microtubules have been detected by transmission electron microscopy in most stages of nuclear division in Naegleria. Their profile length seemed to vary according to the stage of nuclear division and it is argued that this might reflect their function; the long microtubule profiles might represent continuous fibres responsible for the elongation of the nucleus in anaphase-telophase and the short microtubules may be important in transporting chromosomes to the poles i.e. the kinetochore microtubules. Both kinds of microtubule were observed in associations with the poles; the kinetochore microtubules were most evident when the nucleus was in metaphase. By both transmission microscopy and light microscopy, stages in both anaphase A (the moving apart of nuclear material to the daughter cells) and in anaphase B (the elongation of microtubules in order to move the poles apart) could be observed clearly. Colchicine-treatment was used to induce synchronous division in Naegleria cells. During colchicine treatment, Naegleria cells stopped dividing but resumed division almost synchronously ~1 to 2 h after the drug was removed from the medium. Staining of such cells by a-tubulin antibody makes detection of microtubule structures in cells possible. A centrosome with associated microtubules was found by immunofluorescence using anti-tubulin-conjugated antisera to be present in the cytoplasm of Naegleria during nuclear division and this finding contradicts the previous idea that this organelle does not exist in Naegleria. The centrosome appeared to divide during prophase but it could not be detected later at metaphase- telophase. Moreover, transmission electron microscopy did not detect this organelle, though crystalloid structures possibly induced by the colchicine treatment were found near the nuclear envelope in early stages of division. The presence of proteinases in Naegleria spp. and their relation to the life cycle was also investigated. These enzymes were found in all three stages of the amoeba's life cycle; trophozoite, cyst and flagellate. At pH 5.5 in the presence of DTT (dithiothreitol) at least 5 enzymes could be detected in gelatin SDS-PAGE gels for actively multiplying trophozoites; they were named bands A, B, C and DE enzymes . The apparent of band A was -200 kDa, band B was -148 kDa, band C was -116 kDa, band D was -98 kDa and band E was -92 kDa. Bands DE were the most prominent in gels. Flagellates appeared to posses enzymes A and DE, and cysts only band C. Amoebae grown on agar with living bacteria exhibited fewer proteinase bands than those grown in axenic medium; bacterised amoebae lacked bands B and C. The individual proteinases present in Naegleria were characterised on the basis of inhibitor studies, apparent molecular weight, substrate preferences and pH optimum. Band A which was inconsistently observed in gelatin SDS-PAGE gels, had a wide pH range of activity (pH 5.5 to 8.0) but its acitvity was higher at alkaline pH and inhibited by APMSF (4-(amidinophenyl)methanesulphonyl fluoride). Bands B and C were active only at slightly acidic pH (5.5 to 6.0), indicating they are lysosomal in origin; their activity was DTT-dependent, inhibited by E-64 (L-3-carboxy-2,3-trans-epoxypriopionyl-leucylamido-(4-guanidino) butane) and markedly inhibited by antipain, so they could be cysteine proteinases. Doublet DE enzymes, which were membrane-associated, exhibited bimodal activities; at lower pH, their activities were stimulated by DTT but at alkaline pH their activities were higher and not stimulated by DTT. Their activities were inhibited by APMSF, EDTA (ethylenediaminetetraacetic acid) , E-64 and antipain. Band B and C enzymes hydrolysed only the fluorogenic peptidyl-amido methylcoumarin substrate, H-Pro- Phe-Arg-NHMec, in contrast to doublet DE enzymes which hydrolysed substrates H- Pro-Phe-Arg-NHMec, Z-Pro-Arg-NHMec, Bz-Phe-Val-Arg-NHMec and Z-Arg- Arg-NHMec. A similar pattern of proteinases (in SDS-PAGE gelatin gels) was expressed by different stages of growth of amoebae in axenic culture but the intensity of each band appeared to be different and this may have been due to the condition of the trophozoites in cultures during growth. (Abstract shortened by ProQuest.)

A STUDY ON DNA FINGERPRINTING OF AREOLATED GROUPER (Epinephelus areolatus) USING RAPD ANALYSTS

This study reports the application of Random Amplified Polymorphic DNA (RAPD) technique in establishment of DNA fingerprinting of areolated grouper, Epinephelus areolatus, an economically tmportant fish species of the subfamily Epinephelinae (popularly known as grouper

Generation of New Hybridoma UTM-Ha Secreting Monoclonal Antibody Specific to Acanthamoeba species Isolated from Corneal Infection

Acanthamoeba Keratitis is an important corneal infection that caused impaired vision. The Universiti Malaysia Terengganu (UMT) researcher found new species of Acanthamoeba that was being isolated from eye infection in Hospital Kuala Lumpur, which have the characteristic between Acanthamoeba polyphaga and A. castellanii named as HKL-Acantha. This study was aimed to generate specific monoclonal antibody against HKL-Acantha that eventually could be used in diagnosing eye infection among Malaysian. Two Balb/c mice were immunized with sonicated HKL-Acantha through intraperitoneal injection, and anti HKL-acantha IgG, IgM, IgA were measured using ELISA test. Sera from infected mice showed detectable anti-HKL-acantha IgM. The spleen from animal with high antibody titer around (1:81000) was used as a partner in fusion with SP2/0-Ag14 myeloma cells to produce new hybridoma cells, which were then selected and cloned using the selection medium. Two positive hybridoma clones secreting IgM antibodies were obtained and named as hybridoma UTM-Ha1 and UTM-Ha2. Both clones were secreting monoclonal IgM antibody specific to HKL-Acantha. This study suggests the potential of both hybridomas UTM-Ha1 and UTM-Ha2 to generate specific monoclonal IgM against HKL-acantha. This newly generated monoclonal IgM could be used to diagnose the presence of HKL- acantha in patients with eye infection in future

The isolation and identification of Acanthamoeba from air ventilation system samples in selected buildings in Kuantan

Acanthamoeba, a free-living amoeba (FLA) which is abundant in a wide range of the environmental conditions including water, soil and air. Since Acanthamoeba is opportunistic pathogen, some of its species are found to be responsible for serious human diseases. Cysts and trophozoites of Acanthamoeba may be deposited in air ventilation systems with maintaining practices. This could predispose discomfort and diseases to human health especially towards immunosuppressed individuals. Acanthamoeba was isolated from dust samples taken from air ventilation systems in selected buildings in Kuantan. Acanthamoeba was isolated from dust samples taken from air ventilation systems in selected buildings in Kuantan. By using a sterile swab, the dust samples were collected from filters of the air ventilation systems. The dust samples were cultured on the non-nutrient agar plates with inactivate Escherichia coli and incubated in 37ºC incubator. The plates were examined daily for any presence of Acanthamoeba cysts up to 14 days of incubation. The results showed that all 75 dust samples collected were negative. The microscopic examination showed that all non-nutrient agar (NNA) plates were negative for Acantamoeba from day 1 until day 14 of the incubation period. However, fungal growth was observed on the non-nutrient agar (NNA) plates after day 4 of incubation. The negative findings of this study may have been influenced by the environmental factor in Kuantan. Besides that, there are also limitations in the method employed in this study where direct swabs from air ventilation systems was were used instead of using a more specific method such as establishing proper sampling points with air quality instruments to collect air samples. Thus, further studies should consider in order to address these limitations and conditions

Application of PCR-RFLP technique and direct PCR-sequencing analysis to determine the relationships of Acanthamoeba

The genus of Acanthamoeba comprises of several free-living amoebae and mostly found in environment habitats worldwide. These organisms can infect a variety of mammals, including humans involving brain, eyes, skin, bones and lungs (Armstrong, 2000; Martinez, 1999). The taxonomy of Acanthamoeba is has yet to be established and still under review, although species identification of the genus by cyst morphology has been extensively used (Page, 1967)

Genetic relatedness among isolates of Acanthamoeba based on RAPD analysis

Acanthamoeba is a pathogen frequently infecting brain, eyes, skin and lung of human and animal. Seven Acanthamoeba local isolates from various sources, two Acanthamoeba type strains and one strain of Hartmannella vermiformis were characterized for their genetic variability using randomly amplified polymorphic DNA (RAPD) technique using four different 10-mer oligonucleotides primers. Electrophoresis of the amplification products generated DNA bands ranging from approximately 0.25 to 7.50 kbp in size. A genetic relatedness among the isolates was examined using Dice similarity coefficient as the genetic distance measured between the strains of Acanthamoeba and H. vermiformis. Three distinct clusters could be separated at genetic distance of approximately 0.330. © 2006 Asian Network for Scientific Information

Cytotoxicity and genotoxicity studies of synthesized amino acid thiourea derivatives on acanthamoeba spp. and human corneal epithelial cells

Two newly-synthesized amino acids thiourea derivatives; 2-(3-benzoylthioureido)-3-mercaptopropanoic acid and 2-(3-benzoylthioureido)-4-(methylthio)butanoic acid, which were labeled as M1 and M2 respectively, were evaluated for their potential as anti-amoebic agent, aiming for a new discovery in amoebic keratitis treatment. The compounds were tested on Acanthamoeba castellanii (CCAP 1501/2A) and Acanthamoeba sp. (Hospital Kuala Lumpur isolate), and also on human corneal epithelial cells (HCEC). Experiments conducted consisting of IC50 determination by eosin dye and MTT assay, morphological observation by light microscopy, evaluation of membrane integrity by acridine orange/propidium iodide staining, mode of cell death determination by DNA fragmentation test and assessment of DNA damage by alkaline comet assay. The IC50 obtained for M1 were 6.26 μM for A. castellanii, and 9.00 μM for Acanthamoeba sp. (HKL isolate) while for M2 the values were 6.97 and 8.63 μM respectively, indicating that these compounds are cytotoxic against both Acanthamoeba. They shortened acanthopodia structures, transformed the amoeba cells to become rounded, and exhibited no distinct vacuoles and nucleus. The membrane integrity was also disrupted, making them non-intact, and promoted apoptosis in amoeba but did not significantly affected the DNA. Both thiourea derivatives showed moderate cytotoxicity toward HCEC with IC50 at 132.69 and 98.20 μM respectively. The compounds did not significantly alter corneal cells’ cellular morphology. These derivatives were found to disrupt HCEC’s membrane integrity and promoted apoptosis but non-genotoxic on HCEC’s DNA

Cytotoxicity effect of Aaptamine and its derivatives on Acanthamoeba castellanii (IMR isolate)

Acanthamoeba is a free-living amoebae that is ubiquitously present in various natural environments. In this study, several of Aaptamine derivatives (2-5) were synthesized and evaluated for their cytotoxicity effect against Acanthamoeba castellanii (IMR isolate). The Acanthamoeba viability was determine using range of concentration from 0 until 50 µg/mL for each compounds. The treatment was done for 72 hours and Eosin stainning was used to determine the cell viability. From the result obtained, Aaptamine (1) and its derivatives (2-5) have significant effect toward inhibition growth on Acanthamoeba with of 1,4-dibenzylaaptamine (5) was observed as the most potent compound as anti-amoeba agent