Élimination des oeufs de nématodes et des kystes de protozoaires des eaux usées domestiques par lagunage à microphytes en zone soudano-sahélienne

La station expérimentale pilote d'épuration des eaux usées par lagunage de l'École Inter-États d'Ingénieurs de l'Équipement Rural (EIER), Ouagadougou, Burkina Faso, reçoit les eaux usées des bâtiments administratifs et de l'internat des étudiants. Dans les eaux usées brutes, des oeufs d'helminthes (Ascaris lumbricoides, Ankylostoma duodenale), des kystes de protozoaires (Entamoeba coli, Entamoeba histolytica) et des larves d'Anguillule ont été mis en évidence.Avec un temps de séjour de 16,4 jours dans les bassins de lagunage (2 à 3 heures dans le décanteur, 3,4 jours dans le premier bassin, 13 jours dans le deuxième bassin), les kystes d'Entamoeba coli et d' Entamoeba histolytica sont éliminés respectivement à 94 et 96 %, les oeufs d'Ascaris lumbricoides à 100%, les oeufs d'Ankylostoma duodenale à 90 % et les larves d'Anguillule à 92 %. Quand on considère tous les parasites confondus, le décanteur a un rendement éliminatoire de 33 %, le premier bassin 62 % (malgré la forte charge appliquée), le deuxième bassin 78 %. Le rendement global obtenu pour l'ensemble des parasites est de 94 %.Si les évolutions des concentrations des parasites fluctuent dans le temps, il y a une différence très significative entre les concentrations obtenues en sortie du lagunage et celles des eaux usées brutes.On a constaté l'absence des oeufs d'Ascaris lumbricoides pendant toute la période de l'étude ; il en a été de même pour les oeufs d'Ankylostoma duodenale durant une période de 10 mois. Par conséquent, les eaux usées épurées rejetées répondent aux recommandations de l'OMS quant à leur réutilisation agricole.La mise en place des systèmes rustiques d'épuration des eaux usées dans les pays en voie de développement, surtout dans les zones où les ressources en eau sont limitées, pourrait contribuer sensiblement à la diminution des risques sanitaires liés aux pratiques courantes de réutilisation agricole des eaux usées en agriculture.Experimental waste stabilization ponds to purify domestic wastewater from the École Inter-États d'Ingénieurs de l'Équipement Rural (EIER) were built in Ouagadougou, Burkina Faso. The waste stabilization ponds system consists of a raising tank, a decanter, and two ponds in series. The first pond is 1.22 m deep with a surface area of 62 m2; the second pond is 1.07 m deep and covers an area of 340 m2. The inflow averaged 22 m3/d, giving a normal retention time of 3.4 days in pond number 1 and 13 days in pond number 2. The average daily BOD5 load was 1500 kg/ha/day for pond number 1 and 200 kg/ha/day for pond number 2.Different parasites were identified in the raw wastewater, such as cysts of Entamoeba coli and Entamoeba histolytica, eggs of Ascaris lumbricoides and Ankylostoma duodenale and larval stages of Anguillula . The quantitative estimation of cysts, eggs and larvae was obtained using a Mac Master cell after concentration by a formol-ether technique (RITCHIE, 1948). Of 24 samples of raw wastewater analyzed, all contained cysts of E. coli and E. histolytica and Anguillula larvae. Similarly, 100 % of final effluent samples (Fig. 2-B) contained cysts of E. coli and E. histolytica, but the concentrations were respectively 16 and 22 times lower than in raw wastewater. Six of the 24 samples (25 %) contained Anguillula larvae (Fig. 9), 17 % contained Ankylostoma duodenale eggs and no samples (0 %) contained Ascaris lumbricoides eggs (< 1 egg/L).This study were not carried out on Ouagadougou urban wastewater, due to the lack of a wastewater collection network, but the raw wastewater of the central market of Ouagadougou was analyzed for comparison because it represented a larger population than that in the EIER. In the raw wastewater of the central market, the concentration of parasites was high: Ascaris lumbricoides (110 eggs/L), Taenia saginata (53 eggs/L), Ankylostoma duodenale (39 eggs/L), Trichuris trichiura (19 eggs/L), Entamoeba coli (552 cysts/L), Entamoeba histolytica (479 cysts/L), Anguillula (62 larvae/L). The concentration of parasites identified in raw wastewater from EIER (Entamoeba coli (395 cysts/L), Entamoeba histolytica (269 cysts/L), Ascaris lumbricoides (4 eggs/L), Ankylostoma duodenale (9 eggs/L), Anguillula (26 larvae/L)) seemed consistent with the contamination level of the users of EIER stabilization ponds system. With a total retention time of 16.4 days in the stabilization ponds, removals of E. coli and E. histolytica cysts were respectively 94% and 96%. Ascaris lumbricoides and Ankylostoma duodenale eggs were 100 and 90% removed, respectively, and removal of Anguillula larvae was 92%. Grimason et al. (1995), suggest that cumulative retention time of up to 55.3 days at Eldoret, Kenya and 40 days at Meze, France were sufficient to remove between 99.1 % and 99.7 % of cysts of Giardia lamblia, respectively. Global efficiency of the system for all parasites was 94%. In the decanter, where retention time was 2-3 hours, the removal efficiency was 33%. With 3.4 days retention time, the first pond (anaerobic due to the high organic load applied) increases removal to 62%. The second pond (optional) afforded a 78% removal efficiency with a 13 days retention time.If the number of parasites fluctuated with time, the number observed in the effluent was significantly lower than those observed in the influent.The effluent of EIER waste stabilization ponds system was free of Ascaris lumbricoides eggs throughout the study period and free of Ankylostoma duodenale eggs during 10 months; the effluent thus satisfied the WHO recommendations for reuse in agriculture. Waste stabilization pond systems would seem to have a significant place in Sudan- Sahara area for removal of parasite ova and cysts, being economical and advantageous. They would also contribute to reduce sanitary risks associated with reuse of untreated wastewater in agriculture

Effets de la température, du pH et du rayonnement solaire sur la survie de différentes bactéries d'intérêt sanitaire dans une eau usée épurée par lagunage

L'effet du pH (6.2, 7.2, 8.4, 9.6), de la température (4 °C, 12 °C, 23 °C) et du rayonnement solaire a été étudié expérimentalement sur les évolutions des abondances d'Escherichia coli 0126 : B16 et d'autres bactéries pathogènes d'intérêt sanitaire : Salmonella typhimurium et Aeromonas hydrophila. En eau usée épurée, les résultats obtenus montrent que la température et le rayonnement solaire sont parmi les facteurs responsables des variations des abondances de ces bactéries dans les milieux aquatiques. Les faibles valeurs de température (4 °C) favorisent la survie d'E. coli et de S. lyphimurlum et réduisent celle d'A. hydrophila. Les faibles valeurs de température (4 °C) augmentent non seulement la survie bactérienne d'E. coli et de S. typhimurium mais limitent les effets nocifs des pH alcalins (pH 9.6) sur la diminution des abondances de ces bactéries. Parmi les pH alcalins étudiés, le pH 9.6 entraîne la plus forte diminution du temps de survie vis-à-vis d'E. coli, de A. hydrophila et de S. typhimarlam. A pH 9.6, les T90 obtenus à une température de 12 °C sont respectivement de 23, 20 et 33 heures. Le rayonnement solaire joue également un rôle important dans la réduction des abondances bactériennes et ce d'autant plus que le pH est élevé. Dans l'eau usée épurée par lagunage, ajustée à pH 9.6 et exposée au rayonnement solaire, les T90 d'E coli, d'A. hydrophila et de S. typhimarium sont respectivement de 6, 4 et de 6 heures. L'effet combiné du pH et du rayonnement est beaucoup plus important sur la réduction des abondances bactériennes que si l'un des facteurs agit isolément.The behaviour of coliforms especially E. coli, pathogenic bacteria (Salmonella typhimurium), and occasionally pathogenic bacteria (Aeromonas hydrophila) in different aquatic environments has often been studied, but rather than a medical than an environmental point of view. However, studies on the affect of various environmental factors such as temperature, pH and solar radiation on their evolution have seldom been carried out. Their action on pathogenic and occasional pathogenic bacteria remains fragmentary.The aim of this survey is analyse the effect (in laboratory experiment) of certain environmental factors such as temperature, pH and solar radiation on the behaviour of E. coli 0126 : B16, S. typhimurium and A. hydrophila, in waste waters treated in a sewage treatment lagoon. The values of these tested factors are comparable to those which exist in aquatic environments at different seasons.Experimentation has been realized in a variable and complex medium in this case water than the basin of lagoon, in order to study the varying effects of differents factors on evolution of abundance bacteria. Hierarchization of effects of environmental factors or of certain of their associations has been proposed.In order to study the action of the temperature and the combined action of temperature-pH on the bacterial evolution, four sterile 1 litre flasks were filled with 500 ml of waste water treated in lagoon. The pH of each flask was adjusted by acid (H2SO4) or alkaline (NaOH) solutions at 6.2; 7.2; 8.4 and 9.6. Each flask was seeded with a bacterial inoculum of each of the tested strains (E. coli, A. hydrophila and S. typhimurium). The temperature used for the incubation of the flasks corresponded to the different seasons : 4 °C, 12 °C and 23 °C. The temporal evolution of the abundance of each tested strain is followed up by the indirect counting on specific media : TTC and Tergitol Lactose Agar (Institut Pasteur Production) and incubation at 44.5 °C for 24 hours (E. coli), Salmonella-Shigella Agar (BioMérieux) and incubation at 37 °C for 24 hours (S. typhimurium 4,5 H 1,2 i), Pril-Xylose-Ampicilin agar (ROGOL et al., 1979) and incubation at 37 °C for 48 hours (A. hydrophila ATCC 7966).The action of the solar radiation and the combined action of solar radiation-pH on E. coli, S. typhimurium and A. hydrophila was carried out as follows :i) 3 series of glass crystallizers were filled with 500 ml of waste water treated in lagoon (height water : 55 mm). Three reactional mediums were thus prepared by adjusting the pH to 7.2; 8.4 and 9.6. Each pH corresponds to two crystallizers which were seeded with a bacterial inoculum of E. coli; S. typhimurium; and A. hydrophila. For each pH one of the crystallizers is placed in front of a window receiving plenty of solar radiation, the other being protected from all radiation by aluminium paper and used as control. The number of bacteria is verified by counting the colony forming units (c.f.u.) through a dilution-spreading technique on the same media as those used to estimate the action of temperature and pH on the survival of E. coli, A. hydrophila and S. typhimurium.ii) In order to understand the night and day variations in the number of E. coli according to the solar radiation, another method was used. A standard E. coli inoculum is added to some water of the outflow of lagoon filtered on 0,45 µm (Millipore) and exposed to solar radiation. The temporal evolution of the number of E. coli is then followed by daily counts at 6:00 AM, at 2:00 PM and at 19:00 PM (GMT hour).Comparison of the different results is made with T90 (the time needed to reduce the initial bacterial population by 90 %) and with an equality test of 2 regression coefficients (FRONTIER, 1981).Obtained results show that temperature and solar radiation could be considered among factors responsible for the variable abundance of E. coli, A. hydrophila and S. typhimurium. At pH 6.2; 7.2 and 8.4 and effect incubation at 23 °C, the survival time (T90) of E. coli and A. hydrophila is almost the same (T = 53 h) and is anyway inferior to the one for S. typhimurrum which survived nearly twice as long (T90 =100 h). On the other hand, at pH 9.6 and with the same incubation temperature of 23 °C, the survival time of different tested bacteria is reduced. The T90 for E. coli is the same as the one for A. hydrophila (T90 = 20 h) and is 24 hours for S. typhimuriun (table 1). It is almost 4,3 times lower than the one with pH 6.2 and 7.2.The evolution in number of the same bacteria with similar pH but at a temperature of 12 °C are shown in figure 2. Compared with results obtained at 23 °C, one notices that the arrival of different tested bacteria is longer. The survival times for E. coli and A. hydrophila are almost the same (T90 = 73 h), but differ than the one for S. typhimurium (T90 =142 h), a bacteria which always survives better than other bacteria under the same experimental conditions. Low temperatures (4 °C) increase the survival et E. coli and Salmonella typhimurium and reduce that of Aeromonas hydrophila. Not only do these low temperatures (4 °C) reduce survival of E. coli and S. typhimurium but they also reduce negative effects of alkaline pH (pH 9.6) which decreases the abundance of these bacteria. Among the alkaline pH values studied, pH 9.6 caused the highest reduction of the survival of E. coli, A. hydrophila and S. typhimurium.We can conclude that at 4 °C the survival time of A. hydrophila significantly decreases in comparison with that of E. coli and S. typhimurium. At 12 and 23 °C, the survival time of E. coli and A. hydrophila is the same. S. typhimurium survived better than both E. coli and A. hydrophila. Solar radiation is also considered an important factor in the decrease of bacterial abundance especially when the pH is alkaline. Under solar radiation and pH 9.6, the survival time of E. coli, A. hydrophila and S. typhimurium is respectively 6, 4 and 6 hours (table 2). The decline in the numbers of these bacteria in effluent lagoon samples was found to be significantly greater in the presence of both pH-temperature or pH-solar radiation than when each of these factors was acting independently.In waste waters treated in lagoon and under sunlight, E. coli 0126 : B16 undergoes a successive evolution (night growth phase-diurnal decrease phase : fig. 5). According to all count values, we can plot the regression straight line (log 10 bacteria = vs (time)) and estimate the death coefficient which is the slope of this regressional straight line. This model expresses the tendency of the linear decrease of E. coli. Predictions of this model are satisfactory for counting values corresponding to periods of 24 hours or their multiple. The decrease phase or stable night phase cannot be predicte by this model owing to the fact that it is linear and presents the negative slope

Dénombrements directs des bactéries des milieux aquatiques par microscopie en épifluorescence : comparaison entre un système d'analyse d'images automatisé (Mudicam®) et l'observation visuelle

La technique de comptage par microscopie en épitluorescence est la méthode la plus performante pour dénombrer la totalité des bactéries présentes dans les milieux aquatiques. Cependant cette technique est longue, fastidieuse et subjective. Afin d'automatiser et de rendre objectif le dénombrement, le microscope à épifluorescence est couplé à un analyseur d'images. Si les systèmes d'analyse d'images sont utilisés pour les mesures de taille des bactéries aquatiques, très peu d'études font état de comparaison entre les dénombrements par analyse d'image et ceux réalisés de façon traditionnelle. Cet article présente les résultats des dénombrements de souches bactériennes de référence et de bactéries des milieux aquatiques, par la technique de microscopie en épifluorescence des cellules bactériennes marquées au DAPI, réalisés simultanément par observation microscopique visuelle (visuel) et par analyse d'images automatisée (automatique).Le système d'analyse d'images est composé d'une caméra vidéo (Lhesa LH40036) de sensibilité de 510-4 lux, d'une carte de numérisation (512 x 512 pixels, 8 bits, cyclope v 2.32, Digital vision) d'un micro-ordinateur 80-386 et d'un logiciel de dénombrement (Mudicam®. EAU). Le système est couplé à un microscope en épilluorescence Olympus BH2.Les dénombrements ont été réalisés d'une part sur des suspensions de souches bactériennes de référence (n = 30) à différents états physiologiques et sur des échantillons d'eaux (n = 50) d'origines diverses (fleuve, eaux saumâtre, marine et résiduaire). La comparaison des deux méthodes est réalisée par un modèle de régression linéaire et une analyse de variance. Les tests statistiques associés permettent de conclure à une bonne concordance entre les deux méthodes. A partir de l'ensemble des dénombrements réalisés, 18 d'entre eux pris au hasard ont été dénombrés de façon manuelle par deux opérateurs et par le système d'analyse d'image. Il apparaît que les différences de comptage les plus élevées correspondent aux dénombrements effectués par chacun des deux opérateurs. Ceci met en évidence que non seulement le système d'analyse d'image permet une quantification rapide des abondances bactériennes, mais en outre il supprime la subjectivité de l'opérateur tout en réalisant des dénombrements aussi précis.Direct counting by epifluorescence microscopy is the best method available to determine total counts of aquatic bacteria. However, microscopic observation is tedious and time-consuming. A more rapid and certainly less subjective way of counting bacteria is to combine epifluorescence microscopy with an image analysis system. Surprisingly, although image analysis is now a relatively common method to measure the size of aquatic bacteria, very few studies have been devoted to the validation of total counts by image-analysis systems. In this paper, we present data on simultaneous determination of total counts of 4'6-diamidino-2-phenylindole (DAPI) stained bacteria by visual means and by image-analysed (Mudicam® system) epifluorescence microscopy methods.The Olympus microscope BH2 is equipped for epifluorescence with a 100 W Hg lamp and a 100x oil immersion objective (Apo UVFL 160/1.3). The image analysis system consists et a high performance (5 x 10-4 lux) video camera (Lhesa LH40036) and an image processor which digitalizes the video image in a grey scale extending from 0 (black) to 255 (white) into a binary image with 512 x 512 pixels (8 bit, cyclope v 2.32, Digital Vision), and image analysis software (MUDICAM®. EAU). The samples were stained with DAPI (final concentration 2.5 µg/ml) and filtered through polycarbonate inters (0.22µm, Nuclepore Corporation). The surface area of the video image is 76 x 111 µm2.The analysed samples come from culture collections of different bacterial strains (n = 30) submitted to different conditions and incubation times to obtain various physiological states (Table 1). The nature water samples were collected from several aquatic ecosystems : Rhône river, Mediterranean sea, Thau lagoon and Montpellier sewage waters (n = 50). The bacterial abundances ranged from 105 to 108 cells/ml and the size range of the cells varied from 0.63 to 17 µm2. Comparisons between the image analysis and visual counts were made on the basic of thirty fields per filter. The image analysis counts are based on a two step procedure. The video image of each microscopie field is first numerised and stored on a hard disk (153 Mo). When all the fields have been stored, the digitized images are submitted to an automatic thresholding which allows background substraction. Automatic counting of bacterial cells is then performed on the basis of object specifications defined by the operator. These specifications concern the minima and maxima values of the area (expressed in pixel numbers) and the fluorescence (expressed in gray levels) of the objects. The MUDICAM®EAU software also provides the mean number of cells per millilitre and the associated variance.Average concentrations and confidence limits are shown in Table 2 for bacterial collection strain cultures and in Table 3 for water samples. When we compared visual and image analysis counts by- linear regression, the ability of the image analysis system to enumerate bacterial cells was clearly demonstrated. With bacterial culture (Fig. 2) and with water samples (Fig. 3), the coefficients of correlation were respectively r = 0.997 and r = 0.996 (p = 0.0001). The slopes of the models are not significantly different from unity and the Y-intercepts are not different from zero. Moreover we have compared the total visual counts of two experimenters and the image-analysed counts on eighteen random samples (Table 4). The variance analysis shows that there is no difference between the three methods, with mean value of 6.09, 6.08 and 6.11 for the image-analysed method, experimenter n° 1 and experimenter n° 2, respectively. While non significant, the greatest difference in counts was obtained between the two experimenters.If may be concluded that the image analyser tested for total counts by epifluorescence microscopy is a precise and rapid procedure for the determination of total bacterial counts. This method may be standardized and its automation allows the analysis of many samples, an important advantage in ecological studies. Storage of the samples also allows one to treat a posteriori some complementary aspects of the total count, such as the double staining of bacteria. The image analyser tested is appropriate for bacterial ecology studies which require epifluorescence microscopy

Utilisation du bouillon sélénite F modifié pour dénombrer Salmonella dans les milieux aquatiques

Ce travail a pour objet de présenter une nouvelle méthode de dénombrement des Salmonella dans différents types d'eaux (de rivière, saumâtre, eaux usées brutes et épurées) basée sur l'utilisation d'un milieu d'enrichissement au sélénite additionné de Novobiocine et de Pril et sur une adaptation de la technique du N.P.P. à l'ensemencement d'échantillons de grands volumes après filtration permettant de quantifier les très faibles concentrations de Salmonella. Les vérifications des performances de cette méthode d'isolement et de quantification sont basées sur l'étude des croissances de différentes souches de Salmonella et d'autres espèces bactériennes dans le milieu d'enrichissement modifié, ainsi que sur les résultats quantitatifs et qualitatifs fournis par cette méthode lorsqu'elle est appliquée à des échantillons d'eau en provenance de l'environnement aquatique. Ces résultats montrent notamment que la méthode proposée est suffisamment sensible pour détecter 1 à 2 Salmonella dans 10 litres d'eau analysée et qu'elle ne parait pas exclure de sérotypes, du moins parmi ceux les plus fréquemment isolés en France. L'efficacité de la méthode standardisée API Z pour l'identification enzymatique du genre Salmonella a été également testée en référence aux résultats de la sérotypie.This paper presents a new method for enumerating Salmonellae in environmental waters (freshwater, brackishwater, sewage and treated waters) using the F Selenite enrichment broth modified by the addition of Novobiocin and Pril, and an adaptation of the M.P.N. method for the inoculation of large amounts of water after filtration to improve the enumeration of low concentrations of Salmonellae. The verification of the performance of this detection and enumeration method are based on the study of the growth of different Salmonellae species and of others bacterial species in the modified enrichment broth, and on the quantitative and qualitative results obtained by the application of this methodology to aquatic environmental samples. These results show on one hand, that the sensitivity of the proposed method allows to enumerate 1 to 2 Salmonellae in 10 liter samples, and on the other hand that this method to net exclude any serovar from those which are the most frequently isolated in France. The efficiency of the API Z standardized method for the Salmonellae enzymatic identification versus serological identification was also verified

Essais d'épuration des eaux usées de Marrakech par la jacinthe d'eau (Charges organique, bactérienne et parasitologique)

Cette étude est destinée à tester expérimentalement les capacités d'épuration des eaux usées par lagunage à macrophytes (jacinthe d'eau : Eichhornia crassipes), sous les conditions climatiques de Marrakech.L'installation fonctionne en continu avec un débit constant à l'entrée de 10 l/min. La charge admise est de 40 g DCO/M2/j.Sous l'aspect de la production de biomasse végétale, les effluents domestiques constituent un bon substrat nutritionnel. Les taux de croissance et les productions obtenues montrent dans l'ensemble une excellent adaptation d'Eichhornia crassipesà ce milieu. Le maximum de biomasse et de productivité ont été obtenu en période estivale et sont respectivement de: 40 kg MF/m2 et 38,6 MS/m2/j. Il s'est avéré également que la jacinthe d'eau est persistante toute l'année sous le climat méditerranéen aride de Marrakech.L'épuration des eaux usées domestiques par lagunage à macrophyles aboutit à des rendements satisfaisants surtout en période estivale où on obtient un abattement de 87 % de la DCO et une réduction de 95 % des MEST.Sur te plan sanitaire, l'abattement de la charge bactérienne exprimée par les bactéries témoins de contamination fécale peut atteindre jusqu'à 2ULog pour un temps de séjour théorique très court (7 jours).Ce système e par ailleurs fourni des abattement de 100 % des oeufs d'helminthes parasites au niveau de l'eau épurée.The aim of the present study is to experimentaly test the capacities of the mater hyacinth (Eichhornia crassipes) in order to purify wastewater under Manakesh climatic conditions.The experiment was carried al wastewater spreading zone of Marrakesh pretraitement.The experimant's installation is made of two lined water yacinth ponds that receive domestic wastewater.The proposed process is a continuous system with a constant flow rate of 10 l/mn. The theoritical retention time was estimated to 7 days. The allowed load is 40 g COD/m2/day. Macrophytic biomass was observed for both ponds during the experimental period (Match, 1986 - February, 1987). Parameters of organic, bacterial and parasitological loads are studied in order to determine the system efficiency under arid climate.Obtained results show the water hyacinth ability to adapt to Marrakesh climatic conditions. The number of plants doubled at 12 days, this is coherent with results obtained by PENFOUND (1956), BOCK (1969), WESTLAKE (1963, 1975) and SCULTHORPE (1967). Maximum biomass level and productivity were achieved during the summer period : 40 kg WW/m2/day and 38,6 g DW/m2/day respectively. Biomass and productivity obtained under arid climate are similar to results obtained by WOOTEN and DODD (1976), and by DINGES (1976) under subtropical conditions, and higher than chose obtained by JOHN (1985) under temperate climate. The growth period of water hyacinth is estimated to 9 months at Marrakesh, 10 months at subtropical climate (WOLVERTON and MC DONALD, 1976) and limited to 6 months under cold climate (COPELLI et al., 1982; DUBOIS, 1983; SAUZE, 1983; DE CASABIANCA, 1985). Temperature is considered as a limited growth factor of water yacinth. According to FRANCOIS et al. (1977), the water hyacinth growth was stopped when the temperature is lower than 10 °C. Linder Marrakesh arid climate, the temperature is always higher than 10 °C. It was also found that the water hyacinth survive all a year around in the arid climate of Marrakesh.Domestic sewage purification by water hyacinth leads to satisfactory efficiency during the summer concerning reduction of COD: 87 % and TSS : 95 %. This phenomenon may be jointed to the retention time wich was lengthed (9,4 days) in the summer, and the higher biomass density of water hyacinths in this one. The purifying action of floating macrophytes (Eichharnia crassipes) is physical and biological. The root system stabilizes the medium thus favoring sedimentation of TSS and particulate COD both on the bottom of the tank and by trapping in the root hairs. Elimination of COD is realized by means of the action of bacteria which are present, by sedimentation of particulate COD and root filtration.The biological action of the plants is not an important mechanism for COD elimination. The system efficiency is low at the winter and the reduction of COD and TSS have not exceed 60 % and 82 % respectively because the degenering of the water hyacinths.From sanitary point of view, bacterial load reduction expressed by control faecal contamination bacteria achieved 2 log Units for a short theoritical retention time (7 days). This is higher than the result obtained by DUBOIS (1985). Two hypothesis are given to explain reduction of bacterial load by water hyacinths :1) the bacteria are sedimented or trapped in the root hairs of the water hyacinths whith TSS. 2) Water hyacinths may have a capacity to secrete a chemical substance wich could have bactericid or bacteriostatic effect. The improvement of retention time and the addition of one or two supplementary ponds will probably lead to better results. Moreover, this process had also reduced parasitical helminth eggs to undetectable levels (100 %). The parasitical helminth eggs distinguisched at domestic sewage received by the experimental installation, are Taenia, Hymenolepis, Trichuris and Ascaris geints. Their total number vary tram 0 to 120 eggs/l with a mean of 32. Other types of eggs could be encountred generally in waste water as : Toxocara, Oxyure, Capillaria and Taxoascaris (FOX and FITZGERALD,1976) but was not detected by our technique. No helminth eggs were found in purified wastewater descended through water hyacinth ponds. This phenomenon is explained by supposing that the helminth eggs are present in the effluent but it was the detection limit of the employed technique (Bailenger method), or there is no eggs really at the effluent because of their higher specific weight. Ascaris, Taenia and Trichuris eggs have a sedimentation rate of 0,65 m/h, 0,26 m/h and 1,5 m/h respectively (FEACHEM et al.,1983). The eggs sedimented rapidly in the water hyacinths ponds involving a transfer of the effluent pollution to the sediment. Several authors affirmed that the stabilization ponds are an effective means to reduce parasitical helminth eggs of the wastewaters (GLOYNA, 1972; KOWAL, 1985). Hence, if the parasitical risk could be controled in the purified water (effluent), particular attention should be given to sediments. It is also important to point out, that no parasitical nematode is found at the influent. Nematofauna associated to wastewater and roots of water hyacinth, was represented by bacteria consumer nematode. The abundance of nematode is definite by the existence of bacterial food in the wastewaters (CALAWAY, 1963; SHIEMER, 1976). The principal genus determined are Rhabditis sp, Plectus sp. and Mononchoïdes sp. It appears that Rhabditis genus, is dominant in the first pond (94,7 %) of the nematode population. However, the two genus Rhabditis sp. and Plectus sp. are dominant in the second one and represent 50 % and 49 % respectively. The presence of Plectidae in the second basin indicates that is less loaded (ZULINI, 1976). However, under the arid climate conditions of Marrakesh, the process based on water hyacinth for wastewater purification, is faced with two major problems : first, the water loss by evapotranspiration reachs 60 % during the summer time under arid climate of Marrakesh. The development of Mousquito particularly in the summer, constitutes the second problem of our proposed process. Moreover, front economical point of view, the water hyacinths show a good productivity in the summer under arid climate and could be exploited in several field

Elastase Release by Transmigrating Neutrophils Deactivates Endothelial-bound SDF-1α and Attenuates Subsequent T Lymphocyte Transendothelial Migration

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern, and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy, preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell–derived factor-1α (SDF-1α [CXCL12])-mediated T lymphocyte transendothelial migration, without reducing accumulation. In contrast, recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect, which was negated using a specific neutrophil elastase inhibitor. Furthermore, T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase, which selectively cleaved the amino terminus of HUVEC-bound SDF-1α, which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine, ELC (CCL19), and was not reversed by replenishment of SDF-1α, indicating endothelial retention of the inactivated chemokine. In summary, transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors, and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities

Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis

Heparan sulfate (HS) is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF165 binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF165-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS). FGF2- and VEGF165-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF165-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF165-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF165-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation events in FRS2 and VEGFR-2, respectively.These results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells