Biological factors affecting enflagellation of Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeboflagellate that can be evoked to transform from amoebae to flagellates by subculture to nonnutrient buffer. More than half of the amoebae of strains KUL, nN68, and Lovell became enflagellated 300 min after subculture to amoeba-saline, whereas no amoebae of strains NF66, NF69, and HB4 did. N. fowleri nN68 enflagellated best when grown at 32 or 37 degrees C and subcultured to amoeba-saline at 37 or 42 degrees C. Amoebae from the stationary phase of growth enflagellated more readily than did actively growing amoebae. Incubation in expended culture medium from stationary-phase cultures enhanced the capability of growing amoebae to enflagellate after subculture to amoebasaline. Enflagellation was more extensive when the population density in amoebasaline did not exceed 2 x 10(5) amoebae per ml. Cycloheximide at 1 microgram/ml and actinomycin D at 25 micrograms/ml inhibited growth of N. fowleri nN68. Cycloheximide at 0.5 microgram/ml and actinomycin D at 25 micrograms/ml completely prevented enflagellation when added at time zero. Cycloheximide at 0.5 microgram/ml, added 120 to 300 min after initiation of enflagellation, prevented further differentiation and caused existing flagellates to revert to amoeboid cells. Similarly, actinomycin D at 25 micrograms/ml, added 90 to 300 min after initiation of enflagellation, retarded differentiation and caused flagellates to revert. Radiolabeled precursors were incorporated into macromolecules during differentiation in nonnutrient buffer. Enflagellation of N. fowleri is a suitable model for studying regulation of a eucaryotic protist.     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells

The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.     

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.     

Differentiation of Naegleria fowleri and other naegleriae by polymerase chain reaction and hybridization methods

Abstract In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri , since Naegleriae ( N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini ) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, algae, y, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri .     

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim.

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.     

Various methods to assay carbenicillin activity against Pseudomonas aeruginosa]

It was shown that the cultures of Pseudomonas aeruginosa were sensitive to 75-100 micrograms/ml and resistant to 25-50 micrograms/ml of carbenicillin when tested by the method of serial dilutions in the synthetic medium developed by the authors and in Jorgensen's medium. The cultures were incubated for 4 and 16 hours, respectively. The data did not confirm the resistance of the cultures to 25-75 and 100 micrograms/ml carbenicillin when it was determined with the serial dilution method in Pal's medium and the disk diffusion method, respectively. The advantages of the medium developed by the authors for testing P. aeruginosa sensitivity to carbenicillin are discussed.     

High-Resolution Polyacrylamide Gradient Gel Electrophoresis (PGGE) of Isoenzymes from Five Naegleria Species

ABSTRACT. High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis , two N. lovaniensis , one N. jadini , two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.     

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis, two N. lovaniensis, one N. jadini, two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.     

Alterations in chondrocyte morphology, proliferation and binding of 35SO4 due to Fe(III), Fe(II), ferritin and haemoglobin in vitro

Lapine articular chondrocytes in vitro were used to study the effects of Fe3+, Fe2+, ferritin and haemoglobin on cell proliferation, synthesis of proteoglycans and morphological structure. Fe3+ (10, 100 and 500 micrograms/ml) reduced the DNA content of cultures by approximately 35% as well as inhibiting proteoglycan synthesis. Chondrocytes showed positive cytoplasmic staining for both ferric and ferrous ions at the 500 micrograms/ml concentration. Fe2+ (100 micrograms/ml) also decreased DNA content and proteoglycan synthesis, although no iron uptake by the chondrocytes could be detected. Ferritin (1.0, 0.5 and 0.1 micrograms/ml) elicited a significant inhibition of proteoglycan synthesis without affecting cellular DNA synthesis. 1 and 5 micrograms/ml of haemoglobin each reduced the DNA content of cultures by 60%, whilst markedly inhibiting proteoglycan synthesis (75 and 99% respectively). None of the substances tested caused chondrocyte toxicity. The ability of Fe3+, Fe2+, ferritin and, in particular, haemoglobin to inhibit chondrocyte proteoglycan synthesis may represent a pathway whereby cartilage is susceptible to destruction in the haemophilic joint.     

Selective and differential medium for detecting Clostridium botulinum

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.     

Selective and differential medium for detecting Clostridium botulinum.

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Blood plasma fibronectin does not influence fibroblast-induced collagen gel contraction

Human embryo fibroblasts were cultured within three-dimensional collagen gel in the Eagle medium supplemented with 10% calf serum. Addition of fibronectin (Fn) to the nutrient medium neither stimulated nor inhibited gel contraction by fibroblasts. Gel contraction also proceeded in the medium containing ultroser G or Fn exhausted calf serum. Addition of 10 or 50 micrograms/ml of Fn to the medium with ultroser G exerted no effect on the process of gel contraction. Addition of antibodies prepared against Fn (70 micrograms/ml) and tetrapeptide RGDS (100 and 500 micrograms/ml) to the medium supplemented with Fn exhausted calf serum did not change the rate of collagen gel contraction. The results obtained suggest that the rate of collagen gel contraction by fibroblasts is not dependent on the presence of exogenous fibronectin.