Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate.

The free-living amoebae, Acanthamoeba, can act as opportunistic parasites on a wide range of vertebrates and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices, and their ubiquitous distribution. Subgenus classification based on morphology is being replaced by a classification based on the sequences of the 18S rRNA gene with a total of 18 different genotypes (T1-T18). A new environmental strain of Acanthamoeba isolated from a waste water treatment plant is presented in this study as a candidate for the description of the novel genotype T19 after phylogenetic analysis.

Molecular characterization of Acanthamoeba isolated in water treatment plants and comparison with clinical isolates.

A total of 116 samples (44 clinical specimens and 72 environmental samples) have been analyzed for the presence of Acanthamoeba. The environmental samples (ESs) were collected from four drinking water treatment plants (DWTP, n=32), seven wastewater treatment plants (n=28), and six locations of influence (n=12) on four river basins from the central area of Spain (winter-spring 2008). Water samples were concentrated by using the IDEXX Filta-Max(®) system. Acanthamoeba was identified in 65 of the 72 ESs by culture isolation (90.3%) and 63 by real-time PCR (87.5%), resulting in all sampling points (100%) positive for Acanthamoeba when considering both techniques and all the time period analyzed. Nine of the 44 clinical specimens were positive for Acanthamoeba. Seventeen Acanthamoeba strains (eight from four DWTP and nine from clinical samples) were also established in axenic-PYG medium. Twenty-four of the ESs and the 17 Acanthamoeba sp. strains were genotyped as T4/1, T4/8, and T4/9. The eight strains isolated from the DWTP samples were inoculated in nude mouse to ascertain their potential pathogenicity in this model. Animals that were inoculated died or showed central nervous system symptoms 9 days post-inoculation. Examination of immunofluorescence-stained brain and lung tissue sections showed multiple organisms invading both tissues, and re-isolation of throphozoites was successful in these tissues of all infected animals. For the first time, potentially pathogenic Acanthamoeba T4 has been detected in 100% of different types of water samples including tap water and sewage effluents in the central area of Spain suggesting a potential health threat for humans especially for the contact lens wearers.

Vectorial role of Acanthamoeba in Legionella propagation in water for human use.

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

A year-long study of Cryptosporidium species and subtypes in recreational, drinking and wastewater from the central area of Spain.

A year-long longitudinal study was undertaken to evaluate the presence of Cryptosporidium spp. in drinking water treatment plants (DWTPs), wastewater treatment plants (WWTPs) and freshwater bathing beaches (FBBs) from the central area of Spain. Water samples were collected according to USEPA Method 1623, and concentrated by the IDEXX Filta-Max® system. Cryptosporidium species were detected based on PCR-restriction fragment length polymorphism and sequence analyses of the ssuRNA gene. C. hominis and/or C. parvum isolates were subtyped by DNA sequencing of the Gp60 gene. Among 150 samples, 23 (15.3%) were positive by IFAT and 40 (26.7%) by PCR. Cryptosporidium spp. was more frequent in WWTPs (26.2 and 50.8%) and FBBs (12.5 and 17.5%) by IFAT and PCR respectively. Effluent waters from DWTPs were negative for this parasite suggesting that they are suitable for public use. Tertiary treatment in the WWTPs demonstrated a high removal efficiency of Cryptosporidium in the samples evaluated. Cryptosporidium species identified included C. hominis, C. parvum, C. ubiquitum, C. andersoni and C. muris. Subtyping analysis revealed C. hominis IbA10G2 and IeA11G3T3 alleles, which is the first report of the latter in water samples. Cryptosporidium highest frequency was observed in winter and spring. Our data provide information about the occurrence and diversity of Cryptosporidium in water of human use from the central area of Spain.

Degradation of emergent contaminants by UV, UV/H2O2 and neutral photo-Fenton at pilot scale in a domestic wastewater treatment plant.

This study focuses on the removal of 22 selected micropollutants in an effluent from a municipal wastewater treatment plant (MWTP) at pilot scale. A reactor of 37 L with five low pressure mercury lamps emitting at 254 nm (UV254) was used. The 22 micropollutants include 15 pharmaceuticals, 2 X-Ray contrast medias, 1 corrosion inhibitor and 4 biocides/pesticides. Five of these 22 compounds were used as indicative substances as proposed by the Swiss Federal Office for the Environment (FOEN) (carbamazepine, diclofenac, sulfamethoxazole, benzotriazole and mecoprop). Treatments included UV254 light alone, UV254 + H2O2 and UV254 + H2O2+Fe(3+). Wastewater coming from the MWTP already contained iron with an average total iron of 1.6 mg L(-1). Original pH was not modified and remained between 6 and 7. The parameters changed during the experiments to find the optimal conditions were: wastewater flow rate (2-14 m(3) h(-1)), H2O2 concentration (20-50 mg L(-1)) and Fe (III) concentration (0-4 mg L(-1)). Chemicals removal rates were greater than 80% for the majority of the flow rates tested. Operating costs for the different conditions evaluated were also estimated and compared.

A year long study of the presence of free living amoeba in Spain.

Free-living amoeba such as Acanthamoeba and Balamuthia mandrillaris can act as opportunistic parasites on a wide range of vertebrates and they are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices and their ubiquitous distribution. This work was carried out in order to study the presence of these free-living amoebae (FLA) and their possible seasonality in a continental-Mediterranean climate in different types of water. For this purpose, a total of 223 water samples were collected during one year from four drinking water treatment plants (DWTP), seven wastewater treatment plants (WWTP) and six locations of influence (LI) on four river basins from Spain. Water samples were concentrated using the IDEXX Filta-Max(®) system and analyzed by a triplex real time PCR that detects Acanthamoeba, B. mandrillaris and Naegleria fowleri. Agar plates were also seeded for Acanthamoeba culture. From the three FLA studied, N. fowleri was not detected in any sample while B. mandrillaris was found at the entrance of a DWTP; this being, to our knowledge, the first report of these protozoa in water worldwide. On the other hand, the presence of Acanthamoeba observed was higher, 94.6% of the studied points were positive by real time PCR and 85.2% by culture, resulting in 99.1% positive for Acanthamoeba with both methods. All genetically analyzed Acanthamoeba were genotype T4 but nine different T4/DF3 sequences were observed, three of them being described for the first time, assigning new codes. No seasonal distribution of Acanthamoeba was found. These facts should serve as a warning to contact lens wearers of the risk of a poor hygiene when handling their contact lenses. It should also serve as a signal to physicians to consider FLA as a possible causative agent of nervous system infections as well as Acanthamoeba keratitis due to their high environmental presence shown in this study.

Expression of the large I antigen forming beta-1,6-N-acetylglucosaminyltransferase in various tissues of adult mice.

Large I antigen is specifically formed by a beta-1,6-N-acetylglucosaminyltransferase (IGnT), which is a Golgi enzyme. IGnT converts a linear carbohydrate structure, the i antigen, to a branched structure, the I antigen in N-acetyllactosamines. This conversion has been shown to be developmentally regulated in human erythrocytes. In mouse embryonic development, it has been shown that poly-N-acetyllactosamine plays a critical role in the compaction process (Rastan,S., Thorpe,S.J., Scudder,P., Brown,S., Gooi,H.C., and Feizi,T. (1985) J. Embryol. Exp. Morphol., 87, 115-128.). In order to understand the regulation of IGnT expression during mouse development, the IGnT transcripts were studied using in situ hybridization. The cDNA encoding IGnT was isolated from a murine PCC4 teratocarcinoma cDNA library by nucleic acid hybridization using probes generated from the human IGnT cDNA. The IGnT cDNA was used to produce a fusion protein, which was then used as an immunogen to produce polyclonal antibodies against the enzyme. Nucleotide sequence data was used to design oligonucleotide primers and cDNA probes. The primers and probes, antibodies specific to the fusion protein, and previously obtained human anti-I or i sera, were used to analyze adult and embryonic mouse tissues for expression of IGnT and I antigen. To detect IGnT mRNA, in situ reverse-transcription and polymerase chain reaction were performed on tissue sections using the oligonucleotide primers. Amplified DNA products were detected by in situ hybridization using the cDNA probes. IGnT protein was detected by immunohistochemistry using the IGnT fusion-protein antibody. Expression of the carbohydrate structures was detected using human anti-I or i sera. The results presented demonstrate that IGnT and the I antigen appear in epithelial cells and dividing cells. The antigen also appears to be expressed on cells exposed to the lumenal surface of tissues. These results support the conclusions obtained by the previous studies that IGnT and the resultant I antigen may play critical roles during development and in adult organisms.

Glycophorin B and glycophorin E genes arose from the glycophorin A ancestral gene via two duplications during primate evolution.

Human glycophorin A, B, and E genes are homologous from the 5'-flanking region to 1 kilobase downstream from the exon encoding the transmembrane region. Analysis of human Alu sequences at the transition site from the homologous to nonhomologous region suggested that the GPA gene most closely resembles the ancestral gene, whereas GPB and GPE genes arose by homologous recombination within the Alu repetitive sequence, and acquired 3' sequences from an unrelated gene (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623; Kudo, S., and Fukuda, M. (1990) J. Biol. Chem. 265, 1102-1110). To understand glycophorin gene evolution in primate phylogeny, transmembrane and Alu regions of several primate genomes were amplified by the polymerase chain reaction and their sequences were analyzed. These studies revealed that the GPA gene was present in all primates studied, and the GPB gene was present in pygmy chimpanzee, chimpanzee, and gorilla, but absent from orangutan and gibbon. GPE gene was present in all species with a GPB gene, but was detected in only 7 out of 16 gorillas. The 24-base pair insertion sequence found in the transmembrane exon of the human GPE gene was shown to be derived from the ancestral GPB gene and was inserted into the ancestral GPE gene prior to gorilla divergence. The recombination site in the GPA gene was confirmed to be within an Alu repetitive sequence. We conclude that GPB and GPE genes arose from an ancestral GPA gene via two gene duplications occurring during primate evolution, prior to gorilla divergence.

Rheology: how to characterize and to predict the evolution of W/O/W multiple emulsions.

Synopsis The purpose of this work was to compare the evolution of three multiple emulsions (ME) by different controls. The series of water-in-oil-in-water (W/O/W) multiple emulsions were elaborated and controlled by both the usual analyses which permitted the classification of the multiple emulsions with respect to their thermal stability, and the rheological oscillatory and flow analyses which are polyvalent methods. Further, these allow the 'identity card' of the multiple emulsion to be drawn, its evolution to be followed, and moreover, its behaviour being foreseen by simulating ageing by shear. Three different behaviours are observed; a destructuration, a phase inversion and stability. Résumé L'objectif de ce travail est de comparer, à l'aide de différentes analyses, l'evolution de trois émulsions multiples H/L/H. Ces émulsions sont contrôlées par des analyses classiques qui permettent de classer les émulsions en fonction de leur stabilité thermique, et des analyses rhéologiques en dynamique et en écoulement qui permettent de dresser la 'carte d'identité' de l'émulsion multiple, de suivre son évolution et enfin de prévoir son comportement après simulation du vieillissement par le cisaillement. Trois comportements sont observés; une destructuration, une inversion de phase et une stabilité.

Coronary collateral development in swine after coronary artery occlusion.

We have quantified the development of the coronary collateral circulation in the pig. The collateral circulation was induced to grow by placing an ameroid occluder on the left circumflex coronary artery. Two to 16 weeks after ameroid placement, the coronary collateral circulation was identified after the injection of several colors of a silicone polymer into the coronary arteries and the aorta. We identified intercoronary and extracardiac collaterals and quantified their number, location, size, and wall thickness. Intercoronary collaterals grew to a level that represents a 14-fold increase in normal collateral blood flow under resting conditions compared with the values in an animal not subjected to coronary artery occlusion. Extracardiac collaterals could potentially supply approximately 30% of resting flow. The sources of the extracardiac collaterals were the bronchial and internal mammary arteries. Coronary collateral morphometry and DNA synthesis in the pig heart also were examined. Coronary collaterals had significantly less smooth muscle than did normal arterioles. This may account, in part, for the reduced response of the coronary collaterals to vasodilators. We observed intense DNA synthesis in endothelial and smooth muscle cells in the first 2 or 3 weeks of ischemia. However, DNA synthesis rapidly ceased after this time, coincident with coronary collateral reserve values (ischemic/nonischemic regional blood flow ratios during maximal vasodilation) reaching their maximum level. This suggests that failure of the vessels to continue proliferating accounts for the occurrence of the plateau in blood flow levels.