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Gideon, Ajeagah, and Jean E. Karie Mouncharou. "Dynamique de l'abondance des oocystes d'Isospora belli dans un milieu aquatique en zone tropicale (Cameroun)." Hydroécologie Appliquée 20 (January 14, 2015): 85–102. http://dx.doi.org/10.1051/hydro/2014010.
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Une étude visant à isoler, identifier et caractériser les oocystes d'I. belli dans l'Abiergué a été menée d'avril à novembre 2011. Les échantillonnages ont été faits mensuellement dans six stations le long du cours d'eau. Les oocystes ont été identifiés par des techniques de coloration notamment la coloration de Zielh-Neelsen modifiée et celle du Lugol. Les résultats de la biologie révèlent la présence et la distribution des oocystes d'I.belli le long du cours d'eau avec une densité moyenne de 344 oocystes/L. Cette abondance résulte de la contamination par les matières fécales déversées dans l'hydrosystème à partir des latrines-canons. Les oocystes identifiés montrent une prédominance des oocystes à sporoblaste non-individualisé, en moyenne 202 oocystes/L, aussi bien au niveau spatial que saisonnier. Ce travail a mis en évidence la dominance des oocystes de petite taille avec une densité moyenne de 194 oocystes/L dans ce milieu aquatique en zone urbaine. Le calcul du coefficient de corrélation de Spearman montre des corrélations positivement significatives (p = 0,01) entre les nitrates, la turbidité et les oocystes d'une part et une corrélation négativement significative (p = 0,05) entre le CO2 dissous et les oocystes d'autre part.
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Mpoame, M., and J. Tchoumboué. "Elimination périodique des oocystes d’Eimeria chez le poulet pendant la journée en milieu tropical." Revue d’élevage et de médecine vétérinaire des pays tropicaux 49, no. 3 (March 1, 1996): 227–28. http://dx.doi.org/10.19182/remvt.9518.
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Toutes les deux heures de 6 à 18 h, les fèces de 62 poulets de race locale ont été prélevées à Dschang dans l'ouest du Cameroun. La numération des oocystes d'Eimeria dans ces fèces s'est effectuée à l'aide de la cellule de McMaster. Les oocystes étaient les plus abondants à 16 h et 18 h, lorsque le taux d'humidité de l'air était élevé. La période particulière de libération des oocystes est par conséquent considérée comme une adaptation pour la survie et la transmission des coccidies et comme idéale pour le diagnostic.
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Ajeagah, Gideon A., Ellénita Ngoko Kamguep, Moïse Nola, Samuel Foto Menbohan, and Thomas Njine. "Isolement et mise en évidence des oocystes de Cyclospora cayetanensis dans un hydrosystème polysaprobe en zone équatoriale, Afrique Centrale." Revue des sciences de l’eau 27, no. 2 (June 13, 2014): 115–24. http://dx.doi.org/10.7202/1025562ar.
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Peu de données sont disponibles sur la distribution des Apicomplexa parasites du tube digestif de l’homme. Ils causent pourtant des maladies diarrhéiques de très grande envergure. Une étude menée de janvier à août 2011, dans le cours d’eau Olézoa à Yaoundé (Cameroun), a visé à rechercher et caractériser les oocystes de Cyclospora cayetanensis. Les échantillonnages ont été effectués en amont et en aval du cours d’eau, sur deux stations localisées en zone fortement anthropisées. L’identification des oocystes de C. cayetanensis isolés à l’aide de l’observation directe après coloration au Lugol et selon la technique de Ziehl-Neelsen, a montré qu’ils sont constitués de deux sporocystes contenant chacun deux sporozoïtes. La dynamique d’abondance de ce microorganisme est significativement corrélée à la turbidité, à la température et au pH de l’eau (p < 0,01). Par ailleurs, les formes non sporulées sont plus abondantes que les formes sporulées dans l’environnement et sont fortement corrélées aux teneurs en oxygène dissous qui est un paramètre très important pour leur sporogénèse. En amont du cours d’eau, l’abondance la plus élevée de C. cayetanensis a été de 407 oocystes•L-1 et la valeur enregistrées en aval été de 250 oocystes•L-1. La population de C. cayetanensis observée a été largement dominée par les formes non sporulées de taille 8 µm. La concentration des oocystes la plus élevée en utilisant la technique directe et celle de Ziehl-Neelsen a été enregistrée respectivement au cours des mois de juin et juillet; ces périodes se situent respectivement à la fin de la petite saison des pluies et au début de la saison sèche.
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Pichon, G., V. Robert, T. Tchuinkam, B. Mulder, and J. P. Verhave. "Analyse quantitative de la distribution des oocystes dePlasmodium falciparumchezAnopheles gambiae." Parasite 3, no. 2 (June 1996): 161–67. http://dx.doi.org/10.1051/parasite/1996032161.
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Brasseur, P., C. Uguen, A. Moreno-Sabater, L. Favennec, and J. J. Ballet. "Effets des oxydants sur le dékystement des oocystes de Cryptosporidium parvum." Journal européen d’hydrologie 28, no. 2 (1997): 133–39. http://dx.doi.org/10.1051/water/19972802133.
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Bouratbine, A., K. Aoun, R. Barbouche, M. Béjaoui, and R. Ben Ismail. "Fréquent d'isolement des oocystes de Cryptosporidium dans les selles diarrhéiques d'enfants tunisiens." Médecine et Maladies Infectieuses 28, no. 4 (April 1998): 308–10. http://dx.doi.org/10.1016/s0399-077x(98)80056-5.
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NACIRI, M. "La cryptosporidiose. Importance de la contamination de l’eau." INRAE Productions Animales 5, no. 5 (February 29, 1992): 319–27. http://dx.doi.org/10.20870/productions-animales.1992.5.5.4246.
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Cryptosporidium, protozoaire parasite décrit en 1907 par Tyzzer, fut longtemps considéré comme un organisme commensal, rare et spécifique d’hôte. Après 1981, tous les auteurs s’accordèrent à penser que l’espèce C. parvum était commune à tous les mammifères, y compris l’homme, causant des diarrhées néonatales chez les ruminants, des diarrhées à guérison spontanée chez l’homme immunocompétent et des diarrhées cholériformes incurables chez l’homme immunodéficient. La cryptosporidiose était alors considérée comme une zoonose, l’homme s’infectant au contact des animaux malades ou au contact d’animaux de compagnie porteurs sains. Cependant, la cryptosporidiose peut se transmettre directement d’homme à homme comme le démontrent certaines épidémies dans des hôpitaux ou dans des centres aérés, ou indirectement par l’eau de boisson et les aliments souillés. Des recherches effectuées après plusieurs épidémies en Amérique du Nord et au Royaume Uni ont montré que toutes les eaux de surface (lacs, rivières...) sont contaminées par des cryptosporidies. Cette pollution serait principalement due à l’agriculture et à la faune sauvage. Les eaux d’égouts de populations infectées sont aussi source de contamination. Les procédés de traitement des eaux usées, filtration et désinfection, ne sont pas totalement efficaces pour éliminer complètement Cryptosporidium et des oocystes sont retrouvés dans l’eau de boisson. Depuis 1985, les recherches s’intensifient pour mettre au point des techniques fiables afin de déceler la présence des oocystes dans l’eau de boisson, de les identifier et de les inactiver.
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Ould Ahmed Salem, C. B., D. F. Mamadou, and J. Hafid. "Étude de la prévalence des oocystes de Toxoplasma gondii chez le chat à Nouakchott." Bulletin de la Société de pathologie exotique 110, no. 5 (October 20, 2017): 315–17. http://dx.doi.org/10.1007/s13149-017-0577-7.
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Mukiibi-Muka, G., M. O. Otim, G. Musisi, J. Illango, T. Galiwango, and W. Olaho Mukani. "Etude comparative de l’efficacité de l’amprolium et du diclazuril chez des poulets de chair naturellement infectés en Ouganda." Revue d’élevage et de médecine vétérinaire des pays tropicaux 54, no. 1 (January 1, 2001): 33. http://dx.doi.org/10.19182/remvt.9802.
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L'efficacité de deux médicaments anticoccidiens synthétiques (l'amprolium et le diclazuril) a été étudiée chez des poulets de chair naturellement infectés en Ouganda. Les médicaments ont été administrés par voie orale, mélangés dans de l'eau. L'évaluation du niveau d'infection a été effectuée par la numération des oocystes de coccidies. Les deux médicaments ont été efficaces dans le contrôle de l'excrétion des coccidies, mais l'efficacité du diclazuril a été plus grande. L'administration du diclazuril par voie orale au lieu de l'administration habituelle dans la nourriture a été efficace. Aucune différence de poids n'a été observée entre les groupes traités et le groupe témoin jusqu'à l'âge d'abattage.
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Delaunay, A., A. Baishanbo, L. Favennec, and G. Gargala. "Évaluation de la viabilité et de l’infectivité des oocystes de Cryptosporidium par cytométrie en flux." Annales Pharmaceutiques Françaises 62, no. 5 (September 2004): 310–15. http://dx.doi.org/10.1016/s0003-4509(04)94319-2.
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Cox, D., and F. J. L. Robberts. "Improved Cryptosporidium case findings using immunofluorescent microscopy on concentrated stool." African Journal of Clinical and Experimental Microbiology 22, no. 2 (April 8, 2021): 300–303. http://dx.doi.org/10.4314/ajcem.v22i2.25.
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Background: Diarrhoea is a major cause of morbidity in Cape Town, South Africa, and mortality is attributed to a failure to recognize the severity of the condition. Cryptosporidium and Giardia are increasingly recognized as important causes of diarrhoea in Africa however, suboptimal diagnostic techniques may lead to underappreciation of their significance. Our objectives are to compare the diagnostic yield of direct immunofluorescent antigen (DFA) microscopy on concentrated stool samples for Cryptosporidium and Giardia, with the current approach of wet mount microscopy for Giardia and auramine fluorescent stain for Cryptosporidium on unconcentrated stool.Methodology: Stool specimens (n=104) received at our hospital laboratory for routine microbiological investigations were used for the study. Direct wet-mount auramine-phenol fluorescent microscopy (auramine) detection of Cryptosporidium oocysts and wet mount iodine microscopy for Giardia detection, were performed on unconcentrated stool samples, while DFA stain for simultaneous detection of Cryptosporidium and Giardia was performed on sodium-acetate formalin concentrated stool samples. The diagnostic yields of the tests were compared using the MEDCALC® version 18.0Results: Of the 104 stool specimens received for microbiological analysis, only 66 (63.5%) had specific Cryptosporidium requests while 38 (36.5%) had no Cryptosporidium specific requests. Of the 66 specimens, 9 (13.6%) were positive for Cryptosporidium oocysts with DFA while only 1 (1.5%) was positive with auramine staining (p=0.013). The one auramine-positive specimen was also positive by DFA. Auramine stain microscopy gave a sensitivity of 11.1% (95%CI: 0.28-48.25%) and specificity of 100% (95%CI: 93.7%-100%) when compared to DFA. Of the 38 stool specimens without specific Cryptosporidium request, DFA yielded 5 (13.2%) additional positive results. Taken together, Cryptosporidium was detected in 14/104 (13.5%; 95%CI: 8.36–21.7%) specimens and only 1 of 14 (7.1%) specimens with the current routine laboratory testing approach. Giardia was detected by DFA in 3/104 (0.9%) specimens, while direct iodine wet mount microscopy did not yield any positive results (0%). All 3 Giardia-positive specimens had Cryptosporidium oocysts detected by DFA.Conclusion: These data suggest that a large proportion of Cryptosporidium cases remain undetected by the laboratory due to suboptimal testing methods, and failure by clinicians to specifically request for Cryptosporidium detection. There is need to periodically assess the effectiveness of diagnostic microbiology laboratory approaches to diarrhoea, and access to improved diagnostic laboratory techniques will contribute to more accurate differential diagnosis and a broadened understanding of local aetiology of diarrhoea diseases in Africa.
Keywords: Cryptosporidium, Giardia, diarrhoea, stool concentration, DFA, microscopy
French Title: Amélioration des découvertes de cas de Cryptosporidium à l'aide de la microscopie immunofluorescente sur des selles concentrées
Contexte: La diarrhée est une cause majeure de morbidité au Cap, en Afrique du Sud, et la mortalité est attribuée à l'incapacité de reconnaître la gravité de la maladie. Cryptosporidium et Giardia sont de plus en plus reconnus comme des causes importantes de diarrhée en Afrique, cependant, des techniques de diagnostic sous-optimales peuvent conduire à une sous-estimation de leur importance. Nos objectifs sont de comparer le rendement diagnostique de la microscopie à antigène immunofluorescent direct (DFA) sur des échantillons de selles concentrées pour Cryptosporidium et Giardia, avec l'approche actuelle de la microscopie à montage humide pour Giardia et la coloration fluorescente auramine pour Cryptosporidium sur des selles non concentrées.Méthodologie: Des échantillons de selles (n=104) reçus au laboratoire de notre hôpital pour des examens microbiologiques de routine ont été utilisés pour l'étude. La détection directe par microscopie fluorescente auramine-phénol à montage humide (auramine) des oocystes de Cryptosporidium et la microscopie à l'iode à montage humide pour la détection de Giardia, ont été effectuées sur des échantillons de selles non concentrées, tandis que la coloration DFA pour la détection simultanée de Cryptosporidium et de Giardia a été réalisée sur de l'acétate de sodium formaline concentré échantillons de selles. Les rendements diagnostiques des tests ont étécomparés à l'aide de MEDCALC® version 18.0Résultats: Sur les 104 échantillons de selles reçus pour l'analyse microbiologique, seuls 66 (63,5%) avaient des demandes spécifiques de Cryptosporidium tandis que 38 (36,5%) n'avaient pas de demandes spécifiques de Cryptosporidium. Sur les 66 échantillons, 9 (13,6%) étaient positifs pour les oocystes de Cryptosporidium avec DFA tandis que seulement 1 (1,5%) était positif avec coloration à l'auramine (p=0,013). Le seul échantillon positif à l'auramine était également positif au DFA. La microscopie à l'auramine a donné une sensibilité de 11,1% (IC 95%: 0,28-48,25%) et une spécificité de 100% (IC 95%: 93,7% -100%) par rapport au DFA. Sur les 38 échantillons de selles sans demande spécifique de Cryptosporidium, le DFA a donné 5 (13,2%) résultats positifs supplémentaires. Pris ensemble, Cryptosporidium a été détecté dans 14/104 (13,5%; IC à 95%: 8,36–21,7%) et seulement 1 des 14 échantillons (7,1%) avec l'approche actuelle des tests de routine en laboratoire. Giardia a été détecté par DFA dans 3/104 (0,9%) échantillons, tandis que la microscopie directe à l'iode sur monture humide n'a donné aucun résultat positif (0%). Les 3 échantillons positifs à Giardia avaient des oocystes deCryptosporidium détectés par DFA.Conclusion: Ces données suggèrent qu'une grande proportion des cas de Cryptosporidium ne sont pas détectés par le laboratoire en raison de méthodes de test sous-optimales et de l'échec des cliniciens à demander spécifiquement la détection de Cryptosporidium. Il est nécessaire d'évaluer périodiquement l'efficacité des approches de laboratoire de microbiologie diagnostique pour la diarrhée, et l'accès à des techniques de laboratoire de diagnostic améliorées contribuera à un diagnostic différentiel plus précis et à une compréhension élargie de l'étiologie locale des maladies diarrhéiques en Afrique.
Mots clés: Cryptosporidium, Giardia, diarrhée, concentration des selles, DFA, microscopie
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Searcy, Kristin E., Aaron I. Packman, Edward R. Atwill, and Thomas Harter. "Association of Cryptosporidium parvum with Suspended Particles: Impact on Oocyst Sedimentation." Applied and Environmental Microbiology 71, no. 2 (February 2005): 1072–78. http://dx.doi.org/10.1128/aem.71.2.1072-1078.2005.
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ABSTRACT The association of Cryptosporidium parvum oocysts with suspended particles can alter the oocysts' effective physical properties and influence their transport in aquatic systems. To assess this behavior, C. parvum oocysts were mixed with various suspended sediments under a variety of water chemical conditions, and the resulting settling of the oocysts was observed. Direct microscopic observations showed that oocysts attached to suspended sediments. Settling column and batch experiments demonstrated that oocysts are removed from suspension at a much higher rate when associated with sediments. The rate of oocyst sedimentation depended primarily on the type of sediment with which the oocysts were mixed. Changes in background water conditions had a relatively small impact on the extent of oocyst-particle association and the resulting oocyst deposition. We believe that the ubiquitous association of C. parvum oocysts with suspended particles enhances the sedimentation of oocysts in natural waters and that this interaction should generally be considered when predicting the migration of pathogens in the environment.
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Weir, C., G. Vesey, M. Slade, B. Ferrari, D. A. Veal, and K. Williams. "An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts." Clinical Diagnostic Laboratory Immunology 7, no. 5 (September 1, 2000): 745–50. http://dx.doi.org/10.1128/cdli.7.5.745-750.2000.
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ABSTRACT The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidiumoocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies.
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Maho, Angaya, B. S. Toguebaye, and J. Belot. "Coccidies et coccidioses intestinales de la chèvre du Sahel (Hircus reversus) au Sénégal avec la description d'une espèce nouvelle." Revue d’élevage et de médecine vétérinaire des pays tropicaux 50, no. 2 (February 1, 1997): 121–25. http://dx.doi.org/10.19182/remvt.9582.
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Huit espèces d'Eimeria, dont une nouvelle, ont été trouvées chez la chèvre du Sahel (Hircus reversus) au Sénégal : E. arloingi, E. kocharli, E. alijevi, E. ninakohlyakimovae, E. christenseni, E. hirci, E. caprovina et Eimeria panguii n. sp. Les oocystes d'Eimeria panguii n. sp. étaient ovoïdes et dépourvus de capsule micropylaire. Ils mesuraient 48,19 ± 0,73 x 32,45 ± 0,97 m (45-50 x 29-36 m). Le micropyle était étroit. Les résidus oocystiques étaient présents. Les sporocystes étaient ovoïdes, 16,86 ± 0,77 x 8,93 ± 0,34 m (15-18 x 8-10 m). Le corps de Stieda était absent tandis que les résidus sporocystiques étaient présents. Les deux sporozoïtes étaient falciformes et inversés l'un par rapport à l'autre et se recouvraient par leurs deux extrémités. La transmission expérimentale de ces espèces à un chevreau a permis de mettre en évidence l'hypertrophie des cellules de l'épithélium intestinal, la perforation de la muqueuse intestinale et la chute de ses microvillosités.
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Somiya, I., S. Fujii, N. Kishimoto, and R.-H. Kim. "Development of a mathematical model of Cryptosporidium inactivation by ozonation." Water Science and Technology 41, no. 7 (April 1, 2000): 173–80. http://dx.doi.org/10.2166/wst.2000.0130.
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A new mathematical model was developed to express the processes of Cryptosporidium inactivation by ozonation. In this model, five different stages were considered as state variables of Cryptosporidium oocysts for accurate expression of the inactivation. ATP, in vitro excystation and DAPI/PI permeability assays were used to describe the oocyst amounts of different stages. Some reaction constants were estimated by the structure components of oocysts or by the stoichiometry of reactions, while the others were by the oocysts population changes in ozonation. The calculated values of this model were well consistent with experimental inactivation data. Three-log inactivation of sporozoites required about 0.04 mgO3 per unit oocyst (mgC) from the simulation results. Before ozone reacts with sporozoites, more ozone was consumed to oxidize other parts of oocysts and DOC produced. The main path of inactivation of oocysts by ozonation was estimated to be P1 (intact oocysts)→P2 (oocysts with damaged outer oocyst wall)→P4 (oocysts without excystation function)→P5 (oocysts with inactivated sporozoites and no excystation function) from experimental and simulated results.
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Kwa, B. H., M. Moyad, M. A. Pentella, and J. B. Rose. "A Nude Mouse Model as an in vivo Infectivity Assay for Cryptospomdiosis." Water Science and Technology 27, no. 3-4 (February 1, 1993): 65–68. http://dx.doi.org/10.2166/wst.1993.0323.
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Cryptosporidium parvum is an important patliogen of diarrlieal disease which has been implicated in several outbreaks associated with contamination of surface waters. In monitoring for C. parvum in drinking water sources, it is important to asce tain the viability, and more importantly, the infectivity of low numbers of recovered oocysts. Groups of 10 Balb/C nude (nu/nu) mice, 4-8 weeks old at time of inoculation, were infected with C. parvum oocysts from naturally infected calves and purified using Sheather's sucrose gradients. Oocysts were counted using the Merifluor IFA kit (Meridian). Each group of 10 mice were infected with 1,10,100 and 1000 oocysts respectively. Numbers of oocysts per inoculation were determined by limiting dilution, and parallel inocula were counted microscopically to ascertain the accuracy of the dilutions. Two uninfected nude mice were kept in each cage to serve as controls. Mouse stools were collected every 4 days, concentrated using the Fekal Kontrate Concentration Kit (Meridian) and oocysts were counted with a UV microscope using the Merifluor IFA Kit (Meridian). Oocyst counts were expressed in terms of number of oocyst/g feces. Mice inoculated with 1000 oocysts began to shed oocysts on day 32, mice inoculated with 100 oocysts began to shed on days 44-48, mice inoculated with 10 oocysts began to shed on days 56-60, and mice inoculated with 1 oocyst shed on days 68-88. All infected mice continued to shed oocysts intermittently and with variable oocyst counts until day 180 when the experiment was terminated. This study established that it is possible to infect nude mice with very low numbers, down to a single oocyst. We are currently in the process of correlating the nude mouse assay with other viability assays.
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Gale, P. "Risk assessment model for a waterborne outbreak of cryptosporidiosis." Water Science and Technology 41, no. 7 (April 1, 2000): 1–7. http://dx.doi.org/10.2166/wst.2000.0108.
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Counts of Cryptosporidium oocysts in 100L volumes of treated water are simulated for conditions representative of a waterborne outbreak in a surface water-derived supply. Assuming oocysts act independently during infection, the risk of infection is directly related to the arithmetic mean oocyst density in the water supply, which is in turn related to the total number of oocysts which break through treatment. Spatial/temporal heterogeneity of oocyst concentrations in the treated water contributes to monitoring programmes based on “spot-sampling” underestimating the arithmetic mean oocyst density and hence the risk of infection. This could contribute to the reported lack of a clear association between oocyst concentrations measured in drinking water supplies and the risk of waterborne outbreak of cryptosporidiosis in the population. An increase in spatial heterogeneity of oocysts during treatment could also contribute to an overestimation of the net oocyst removal by treatment.
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Jenkins, Michael B., Barbara S. Eaglesham, Larry C. Anthony, Scott C. Kachlany, Dwight D. Bowman, and William C. Ghiorse. "Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 76, no. 6 (January 22, 2010): 1926–34. http://dx.doi.org/10.1128/aem.02295-09.
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ABSTRACT The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.
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Butkus, Michael A., J. Timothy Bays, and Michael P. Labare. "Influence of Surface Characteristics on the Stability of Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 69, no. 7 (July 2003): 3819–25. http://dx.doi.org/10.1128/aem.69.7.3819-3825.2003.
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ABSTRACT Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.
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Stadterman, K. L., A. M. Sninsky, J. L. Sykora, and W. Jakubowskii. "Removal and inactivation of cryptosporidium oocysts by activated sludge treatment and anaerobic digestion." Water Science and Technology 31, no. 5-6 (March 1, 1995): 97–104. http://dx.doi.org/10.2166/wst.1995.0572.
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To determine the fate of Cryptosporidium parvum oocysts during wastewater treatment, a model of an activated sludge treatment plant was designed with a flow of 17 ml/min and a detention time of 6 hours. Samples of raw sewage were seeded with oocysts and primary and secondary effluents were analyzed for C. parvum using an immunofluorescent technique. To compare removal efficiencies of oocysts by various wastewater treatment processes, raw sewage, activated sludge, trickling filter and biodisc effluents were seeded with oocysts and settled for 2 hr and for the respective detention times. Sludge produced by a wastewater treatment plant and anaerobically digested at 37° C in a laboratory digester was also seeded with C. parvum oocysts. Oocyst inactivation was measured by excystation and direct counts. Removal of oocysts in primary and secondary sedimentation averaged 83.4% and 90.7% respectively. The total oocyst removal in sewage treatment averaged 98.6%. In comparison with other treatment processes, activated sludge had the maximum oocyst removal efficiency at 92%. The anaerobic digestion process inactivated 90% of the oocysts within four hours of exposure. 99.9% of the oocysts were eliminated by anaerobic digestion after 24 hours. This demonstrates that the activated sludge process and anaerobic digestion can be effective for the removal and inactivation of C. parvum oocysts.
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Sturbaum, Gregory D., Carrie Reed, Paul J. Hoover, B. Helen Jost, Marilyn M. Marshall, and Charles R. Sterling. "Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2665–68. http://dx.doi.org/10.1128/aem.67.6.2665-2668.2001.
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ABSTRACT Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum,Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguishedC. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolatedC. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.
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Gale, P. "Simulating cryptosporidium exposures in drinking water during an outbreak." Water Science and Technology 38, no. 12 (December 1, 1998): 7–13. http://dx.doi.org/10.2166/wst.1998.0486.
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This paper predicts exposures to Cryptosporidium parvum oocysts through drinking water under conditions which are consistent with a waterborne outbreak. Sources of variation which contribute to the variation in oocyst exposures include the oocyst densities in the raw waters, the efficiency of oocyst removal by treatment and the daily consumption of unboiled tap water. Even under outbreak conditions the majority of consumers may not ingest any oocysts each day. Of those who are exposed, some ingest just one oocyst/d while others ingest higher doses, which in a small proportion approach the ID50 for C parvum. Ignoring this variation and using a single point average exposure predicts that a much larger proportion of the population is exposed each day but only ever to very low doses of oocysts. The impact of ignoring this variation on the predicted risks depends on the nature of the dose-response curve and, in particular, the assumptions made about the low dose extrapolation. The heterogeneity of oocyst densities in drinking water during an outbreak could contribute to the failure to detect oocysts in some waterborne outbreaks.
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DENG, MING QI, and DEAN O. CLIVER. "Inactivation of Cryptosporidium parvum Oocysts in Cider by Flash Pasteurization." Journal of Food Protection 64, no. 4 (April 1, 2001): 523–27. http://dx.doi.org/10.4315/0362-028x-64.4.523.
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Cryptosporidium parvum is a well-recognized pathogen of significant medical importance, and cider (apple juice) has been associated with foodborne cryptosporidiosis. This study investigated the effect of flash pasteurization on the viability of contaminant C. parvum oocysts. Cider inoculated with oocysts was heated at 70 or 71.7°C for 5, 10, or 20 s, and oocyst viability was measured by a semiquantitative in vitro infectivity assay. By infecting multiple wells of confluent Madin-Darby bovine kidney cells with serial dilutions of heat-treated oocysts and examining infected cells by indirect fluorescent antibody staining, the most probable number technique was applied to quantify log reduction of oocyst viability. Heating for 10 or 20 s at either temperature caused oocyst killing of at least 4.9 log (or 99.999%), whereas oocyst inactivation after pasteurization for 5 s at 70 and 71.7°C was 3.0 log (99.9%) and 4.8 log (99.998%), respectively. Our results suggested that current practices of flash pasteurization in the juice industry are sufficient in inactivating contaminant oocysts.
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NICHOLS, R. A. B., C. A. PATON, and H. V. SMITH. "Survival of Cryptosporidium parvum Oocysts after Prolonged Exposure to Still Natural Mineral Waters." Journal of Food Protection 67, no. 3 (March 1, 2004): 517–23. http://dx.doi.org/10.4315/0362-028x-67.3.517.
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The survival kinetics of purified Cryptosporidium parvum oocysts of both human and ovine origin, immersed in four still natural mineral waters (total dissolved salts ranging from 91 mg/liter to 430 mg/liter) and reverse osmosis water was assessed by inclusion or exclusion of the fluorogenic vital dyes 4′,6-diamidino-2-phenylindole and propidium iodide over a 12-week period. Semipermeable chambers were used to contain the oocysts while immersed in each mineral water type, permitting both intimate interactions between oocysts and matrices and straightforward sampling for viability assessments. The viability of both oocyst types, assessed at weekly intervals, remained unaltered after 12 weeks at 4°C, whereas a progressive decline in the viability of both oocyst isolates was observed when immersed in mineral waters at 20°C. At 20°C, approximately 30% of oocysts remained viable after 12 weeks incubation. Here, temperature was the major factor that adversely affected oocyst survival, although higher mineral content was also proportionally and significantly associated with this increased oocyst inactivation. The prolonged survival of oocysts at 4°C in our studies indicates that they could survive for prolonged periods of time in U.K. groundwaters (average temperature ~10°C) and thus represent a potential public health hazard if contamination of mineral water sources by viable oocysts were to occur.
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Wolyniak, E. A., B. R. Hargreaves, and K. L. Jellison. "Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory." Applied and Environmental Microbiology 76, no. 4 (December 18, 2009): 1021–27. http://dx.doi.org/10.1128/aem.01804-09.
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ABSTRACT Cryptosporidium is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of C. parvum oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics.
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LE GOFF, L., B. HUBERT, L. FAVENNEC, I. VILLENA, J. J. BALLET, A. AGOULON, N. ORANGE, and G. GARGALA. "Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice." Journal of Food Protection 78, no. 12 (December 1, 2015): 2247–52. http://dx.doi.org/10.4315/0362-028x.jfp-15-062.
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Cryptosporidium spp., a significant cause of foodborne infection, have been shown to be resistant to most chemical food disinfectant agents and infective for weeks in irrigation waters and stored fresh vegetal produce. Pulsed UV light (PL) has the potential to inactivate Cryptosporidium spp. on surfaces of raw or minimally processed foods or both. The present study aimed to evaluate the efficacy of PL on viability and in vivo infectivity of Cryptosporidium parvum oocysts present on raspberries, a known source of transmission to humans of oocyst-forming apicomplexan pathogens. The skin of each of 20 raspberries was experimentally inoculated with five 10-μl spots of an oocyst suspension containing 6 × 107 oocysts per ml (Nouzilly isolate). Raspberries were irradiated by PL flashes (4 J/cm2 of total fluence). This dose did not affect colorimetric or organoleptic characteristics of fruits. After immunomagnetic separation from raspberries, oocysts were bleached and administered orally to neonatal suckling mice. Seven days after infection, mice were euthanized, and the number of oocysts in the entire small intestine was individually assessed by immunofluorescence flow cytometry. Three of 12 and 12 of 12 inoculated mice that received 10 and 100 oocysts isolated from nonirradiated raspberries, respectively, were found infected. Four of 12 and 2 of 12 inoculated mice that received 103 and 104 oocysts from irradiated raspberries, respectively, were found infected. Oocyst counts were lower in animals inoculated with 103 and 104 oocysts from irradiated raspberries (92 ± 144 and 38 ± 82, respectively) than in animals infected with 100 oocysts from nonirradiated raspberries (35,785 ± 66,221, P = 0.008). PL irradiation achieved oocyst reductions of 2 and 3 log for an inoculum of 103 and 104 oocysts, respectively. The present pilot-scale evaluation suggests that PL is an effective mode of decontamination for raspberries and prompts further applicability studies in industrial contexts.
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BUKHARI, Z., and H. V. SMITH. "SHORT PAPER Cryptosporidium parvum: oocyst excretion and viability patterns in experimentally infected lambs." Epidemiology and Infection 119, no. 1 (August 1997): 105–8. http://dx.doi.org/10.1017/s0950268897007590.
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Cryptosporidium parvum infections of domestic animals can have a considerable economic impact and as oocysts are voided in the faeces of infected hosts, environmental contamination with agricultural waste has also become a matter of concern. Since only viable oocysts are potentially infectious, the numbers of oocysts excreted during infection can have important implications for both veterinary and public health. During the course of infection in experimentally infected lambs, oocyst viability was assessed by a fluorogenic vital dyes assay and by a maximized in vitro excystation assay. The excreted oocyst populations contained a higher proportion of viable oocysts 5–11 days post infection (d.p.i.) than later in the infection. Oocyst viability declined consistently 11–15 d.p.i. and coincided with periods when peaks in serum and intestinal anti-Cryptosporidium antibodies have been reported to occur. Infected lambs excreted a mean of 4·8 (standard error [S.E.]±0·4)×109 oocysts per g of faeces, of which half were non-viable and therefore of no significance for disease transmission. This study demonstrates that the numbers of viable oocysts excreted by infected lambs is smaller than previously suspected.
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Inomata, A., N. Kishida, T. Momoda, M. Akiba, S. Izumiyama, K. Yagita, and T. Endo. "Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid and high-sensitive detection of Cryptosporidium in water samples." Water Science and Technology 60, no. 8 (October 1, 2009): 2167–72. http://dx.doi.org/10.2166/wst.2009.599.
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We describe a novel assay for simple, rapid and high-sensitive detection of Cryptosporidium oocysts in water samples using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The detection limit of the developed RT-LAMP assay was as low as 6 × 10−3 oocysts/test tube, which theoretically enables us to detect a Cryptosporidium oocyst and perform duplicated tests even if water samples contain only one oocyst. The developed RT-LAMP assay could more sensitively detect Cryptosporidium oocysts in real water samples than the conventional assay based on microscopic observation.
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NICHOLS, ROSELY A. B., and HUW V. SMITH. "Optimization of DNA Extraction and Molecular Detection of Cryptosporidium Oocysts in Natural Mineral Water Sources." Journal of Food Protection 67, no. 3 (March 1, 2004): 524–32. http://dx.doi.org/10.4315/0362-028x-67.3.524.
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The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65°C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65°C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.
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Guyot, K., M. F. Gireaudot-Liepmann, A. Cabon, I. Riveau-Ricard, M. Lange, J. M. Delattre, and E. Dei-Cas. "Influence of US EPA 1622 method successive steps on the viability of Cryptosporidium oocysts." Water Science and Technology 41, no. 7 (April 1, 2000): 189–96. http://dx.doi.org/10.2166/wst.2000.0132.
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Viable Cryptosporidium parvum oocysts were processed by the US EPA 1622 method to determine if the procedure that requires successive filtration, elutionand centrifugation alters their integrity and viability (determined by in vitro excystation). Oocyst seeded in tap water samples were also used to evaluate recovery efficiencies and impact of the whole procedure on oocyst viability. Filtration through Envirochek Gelman cartridge was found not to damage oocysts. The use of Laureth-12 buffer during the elution step was shown to lead to greater spontaneous oocysts excystation than other phosphate buffers containing between 80 and/or SDS (like the Gelman buffer). However, this drawback was widely balanced against the best efficiency of this buffer to elute oocysts captured by the cartridge filter and therefore against its high recovery efficiency. Thus, in water samples in which the oocyst concentration is expected to be low, it is more advantageous to employ the Laureth-12 buffer for the elution through it can influence viability. Centrifugation speeds (1,000–5,000 g) did not alter oocysts.
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Chauret, Christian, Kerry Nolan, Ping Chen, Susan Springthorpe, and Syed Sattar. "Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine." Canadian Journal of Microbiology 44, no. 12 (December 1, 1998): 1154–60. http://dx.doi.org/10.1139/w98-113.
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Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2-µm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3; pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log10·day-1) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.Key words: Cryptosporidium, survival, disinfection, biological antagonism.
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Kuczynska, Ewa, Daniel R. Shelton, and Yakov Pachepsky. "Effect of Bovine Manure on Cryptosporidium parvum Oocyst Attachment to Soil." Applied and Environmental Microbiology 71, no. 10 (October 2005): 6394–97. http://dx.doi.org/10.1128/aem.71.10.6394-6397.2005.
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ABSTRACT The objective of this work was to assess the effect of dilute bovine manure (1.0% and 0.1%) versus that of no manure on attachment and subsequent detachment of Cryptosporidium parvum oocysts to soil. Manure enhanced the attachment of oocysts to soil particles; the maximum attachment was observed with 0.1% manure. Oocyst attachment was partially reversible; maximum detachment was observed with dilute manure. These results indicate that oocyst attachment to soil is substantially affected by bovine manure in a complex manner and should have implications for how oocysts may be transported through or over soils.
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KNIEL, K. E., and M. C. JENKINS. "Detection of Cryptosporidium parvum Oocysts on Fresh Vegetables and Herbs Using Antibodies Specific for a Cryptosporidium parvum Viral Antigen." Journal of Food Protection 68, no. 5 (May 1, 2005): 1093–96. http://dx.doi.org/10.4315/0362-028x-68.5.1093.
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The purpose of this study was to determine if the viral symbiont of Cryptosporidium parvum (CPV) sporozoites could be used as a target for sensitive detection of the parasite in food samples. Polyclonal sera specific to the recombinant viral capsid protein (rCPV40) was used in a dot blot hybridization assay to detect oocysts recovered from green onions and cilantro. Small batches of chopped green onions and cilantro leaves were artificially contaminated with three different concentrations of oocysts: 106, 102, and 101. rCPV40 was superior in detecting oocysts compared with other antibodies directed toward total oocyst protein and oocyst surface antigens. This study provides evidence that CPV is an excellent target for sensitive detection of C. parvum oocysts in foods.
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Pezzana, A., Ph Vilaginès, F. Bordet, D. Coquard, B. Sarrette, and R. Vilaginès. "Optimization of the Envirochek capsule method and immunomagnetic separation procedure for the detection of low levels of Cryptosporidium in large drinking water samples." Water Science and Technology 41, no. 7 (April 1, 2000): 111–17. http://dx.doi.org/10.2166/wst.2000.0122.
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The method for concentration of Cryptosporidium oocysts in large drinking water samples using the Envirocheck capsule has been optimized for the detection of low levels of oocysts. Elution from the filter by contact time and vortex agitation gave 68% oocyst recovery. Centrifugation (1,250 g; 30 min; 4°C) improved recovery to 94% without morphological damage of the oocysts. Increasing the ratio of magnetic beads to sample volume in the IMS procedure led to 69% efficiency. In these conditions, the overall recovery of the procedure was 49% as assessed with low oocysts spike doses in 100 litres tap water samples. The methodology described allows the detection of 0.1 oocyst per litre when 100 litres samples are processed.
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Delaunay, Agnès, Gilles Gargala, Xunde Li, Loic Favennec, and Jean Jacques Ballet. "Quantitative Flow Cytometric Evaluation of MaximalCryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model." Applied and Environmental Microbiology 66, no. 10 (October 1, 2000): 4315–17. http://dx.doi.org/10.1128/aem.66.10.4315-4317.2000.
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ABSTRACT The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.
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Smith, H. V., B. M. Campbell, C. A. Paton, and R. A. B. Nichols. "Significance of Enhanced Morphological Detection of Cryptosporidium sp. Oocysts in Water Concentrates Determined by Using 4′,6′-Diamidino-2-Phenylindole and Immunofluorescence Microscopy." Applied and Environmental Microbiology 68, no. 10 (October 2002): 5198–201. http://dx.doi.org/10.1128/aem.68.10.5198-5201.2002.
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ABSTRACT Of 2,361 water concentrates analyzed for the presence of Cryptosporidium spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-Cryptosporidium sp. monoclonal antibody and the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.
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Wolyniak DiCesare, E. A., B. R. Hargreaves, and K. L. Jellison. "Biofilm Roughness Determines Cryptosporidium parvum Retention in Environmental Biofilms." Applied and Environmental Microbiology 78, no. 12 (April 6, 2012): 4187–93. http://dx.doi.org/10.1128/aem.08026-11.
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ABSTRACTThe genusCryptosporidiumis a group of waterborne protozoan parasites that have been implicated in significant outbreaks of gastrointestinal infections throughout the world. Biofilms trap these pathogens and can contaminate water supplies through subsequent release. Biofilm microbial assemblages were collected seasonally from three streams in eastern Pennsylvania and used to grow biofilms in laboratory microcosms. Daily oocyst counts in the influx and efflux flow allowed the calculation of daily oocyst retention in the biofilm. Following the removal of oocysts from the influx water, oocyst attachment to the biofilm declined to an equilibrium state within 5 days that was sustained for at least 25 days. Varying the oocyst loading rate for the system showed that biofilm retention could be saturated, suggesting that discrete binding sites determined the maximum number of oocysts retained. Oocyst retention varied seasonally but was consistent across all three sites; however, seasonal oocyst retention was not consistent across years at the same site. No correlation between oocyst attachment and any measured water quality parameter was found. However, oocyst retention was strongly correlated with biofilm surface roughness and roughness varied among seasons and across years. We hypothesize that biofilm roughness and oocyst retention are dependent on environmentally driven changes in the biofilm community rather than directly on water quality conditions. It is important to understand oocyst transport dynamics to reduce risks of human infection. Better understanding of factors controlling biofilm retention of oocysts should improve our understanding of oocyst transport at different scales.
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Nichols, Rosely A. B., Brian M. Campbell, and Huw V. Smith. "Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring." Applied and Environmental Microbiology 72, no. 8 (August 2006): 5428–35. http://dx.doi.org/10.1128/aem.02906-05.
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ABSTRACT We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.
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Jenkins, M. B., M. J. Walker, D. D. Bowman, L. C. Anthony, and W. C. Ghiorse. "Use of a Sentinel System for Field Measurements ofCryptosporidium parvum Oocyst Inactivation in Soil and Animal Waste." Applied and Environmental Microbiology 65, no. 5 (May 1, 1999): 1998–2005. http://dx.doi.org/10.1128/aem.65.5.1998-2005.1999.
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ABSTRACT A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purifiedCryptosporidium parvum oocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50°C and decreases in soil water potential (−0.003 to −3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect of soil freeze-thaw cycles was evident in the large proportion of empty sentinel oocysts. The potentially infective sentinel oocysts were reduced to <1% while the proportions in controls did not decrease below 50% potentially infective during the first field experiment. Microscopic observations of empty oocyst fragments indicated that abrasive effects of soil particles were a factor in oocyst inactivation. A similar pattern was observed in a second field experiment at the same site.
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Atwill, E. R., B. Hoar, M. das Graças Cabral Pereira, K. W. Tate, F. Rulofson, and G. Nader. "Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding of Cryptosporidium parvum in Adult Beef Cattle." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4604–10. http://dx.doi.org/10.1128/aem.69.8.4604-4610.2003.
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ABSTRACT Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding of C. parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces−1, with a 54% probability of detecting just one oocyst g of feces−1. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces−1. The estimated environmental loading rate of C. parvum ranged from 3,900 to 9,200 oocysts cow−1 day−1, which is substantially less than a previous estimate of 1.7 × 105 oocysts cow−1 day−1 (range of 7.7 × 104 to 2.3 × 105 oocysts cow−1 day−1) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.
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Slifko, Theresa R., Debra E. Huffman, and Joan B. Rose. "A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts." Applied and Environmental Microbiology 65, no. 9 (September 1, 1999): 3936–41. http://dx.doi.org/10.1128/aem.65.9.3936-3941.1999.
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ABSTRACT Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed Pvalue (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log10 inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.
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Sekikawa, Takahiro. "A new immunomagnetic bead separation–surfactant extraction treatment protocol for rapid and sensitive quantitative PCR detection of Cryptosporidium parvum DNA." Water Supply 17, no. 1 (July 27, 2016): 161–68. http://dx.doi.org/10.2166/ws.2016.125.
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The Cryptosporidium oocyst is encased in a robust wall that is extremely resistant to detrimental environmental factors such as chlorine used to disinfect potable water. Therefore, extracting oocyst DNA is not a trivial undertaking. Standard procedures used to extract DNA from oocysts, such as freeze–thaw (F/T) methods and DNA purification kits, are time-consuming and expensive and are difficult to implement in routine clinical practice. Therefore, we developed a surfactant extraction treatment (SET) that efficiently extracts DNA from the oocyst. Immunomagnetic separation (IMS) combined with quantitative real-time polymerase chain reaction (qPCR) detects pathogenic microorganisms with high sensitivity. The objective of the present study was to evaluate SET for its ability to simplify qPCR detection of 18S rDNA directly from immunomagnetic bead–oocyst conjugates. DNA extracted directly from the conjugates using SET did not affect DNA amplification in the qPCR assay. Further, the rate of DNA amplification using IMS–SET was greater than that using F/T combined with the DNA purification kit. The rate of recovery of oocysts from surface water samples spiked with oocysts did not differ significantly from previously published values. These data demonstrate that the new IMS–SET protocol using qPCR can simplify the recovery and detection of Cryptosporidium oocysts.
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Bumstead, N., and B. J. Millard. "Variation in susceptibility of inbred lines of chickens to seven species ofEimeria." Parasitology 104, no. 3 (June 1992): 407–13. http://dx.doi.org/10.1017/s0031182000063654.
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The pattern of oocyst production of 8 inbred lines of chickens was compared for each of the 7 species ofEimeriawhich infect this host. Both the overall numbers and the pattern of oocyst production differed in the inbred lines, but there was no evidence of prolonged cycling of schizogenic developmental stages. Comparison of the numbers of oocysts produced by the different lines indicates that there may be common genetic factors affecting susceptibility to 6 of the 7 species. Surprisingly there appears to be an inverse relationship between susceptibility toE. tenellaand susceptibility to the other species: lines which produced most oocysts ofE. tenellaproduced least oocysts of the other species andvice-versa.
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Al-Adhami, B. H., R. A. B. Nichols, J. R. Kusel, J. O'Grady, and H. V. Smith. "Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy." Applied and Environmental Microbiology 73, no. 3 (September 29, 2006): 947–55. http://dx.doi.org/10.1128/aem.01251-06.
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ABSTRACT To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ�cm−2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ�cm−2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ�cm−2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ�cm−2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.
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Kaucner, C., C. M. Davies, C. M. Ferguson, and N. J. Ashbolt. "Evidence for the existence of Cryptosporidium oocysts as single entities in surface runoff." Water Science and Technology 52, no. 8 (October 1, 2005): 199–204. http://dx.doi.org/10.2166/wst.2005.0264.
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There is uncertainty whether Cryptosporidium oocysts attach to particles or to each other under ambient water conditions. Particle size distributions of Cryptosporidium oocyst suspensions were determined over a range of ionic strengths and pHs to determine under those environmental conditions that may promote oocyst aggregation. Cryptosporidium oocysts were shown to only aggregate in high ionic strength solutions (>0.45 M) and remain largely as single entities at ionic strengths and pHs that were likely to be encountered in surface runoff. Similarly, in loam soil suspensions, rather than attaching to the soil particles the majority of oocysts also remained as single entities. Overall, oocysts are expected to remain largely unattached to either themselves or soil particles in overland runoff. This has implications for pathogen transport and modelling since oocysts that are freely suspended are more likely to be transported in runoff to surface waters than if attached to more dense soil/faecal particles.
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KNIEL, KALMIA E., ADRIENNE E. H. SHEARER, JENNIFER L. CASCARINO, GARY C. WILKINS, and MARK C. JENKINS. "High Hydrostatic Pressure and UV Light Treatment of Produce Contaminated with Eimeria acervulina as a Cyclospora cayetanensis Surrogate." Journal of Food Protection 70, no. 12 (December 1, 2007): 2837–42. http://dx.doi.org/10.4315/0362-028x-70.12.2837.
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The prevalence, size, genome, and life cycle of Eimeria acervulina make this organism a good surrogate for Cyclospora cayetanensis, a protozoan that causes gastroenteritis in humans, including recent outbreaks in the United States and Canada associated with contaminated raspberries and basil. Laboratory studies of C. cayetanensis are difficult because of the lack of readily available oocysts and of infection models and assays. UV radiation and high-hydrostatic-pressure processing (HPP) are both safe technologies with potential for use on fresh produce. Raspberries and basil were inoculated with sporulated E. acervulina oocysts at high (106 oocysts) and low (104 oocysts) levels, and inoculated and control produce were treated with UV (up to 261 mW/cm2) or HPP (550 MPa at 40°C for 2 min). Oocysts recovered from produce were fed to 3-week-old broiler chickens, which were scored for weight gain, oocyst shedding, and lesions at 6 days postinoculation. Oocysts exhibited enhanced excystation on raspberries but not on basil. Birds fed oocysts from UV-treated raspberries had reduced infection rates, which varied with oocyst inoculum level and UV intensity. Birds fed oocysts from UV-treated raspberries (104 oocysts) were asymptomatic but shed oocysts, and birds fed oocysts from UV-treated basil (104 oocysts) were asymptomatic and did not shed oocysts. Birds fed oocysts from HPP-treated raspberries and basil were asymptomatic and did not shed oocysts. These results suggest that UV radiation and HPP may be used to reduce the risk for cyclosporiasis infection associated with produce. Both treatments yielded healthy animals; however, HPP was more effective, as indicated by results for produce with higher contamination levels.
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Machado, E. C. L., T. L. M. Stamford, L. C. Alves, R. G. Melo, and N. K. S. Shinohara. "Effectiveness of Cryptosporidium spp. oocysts detection and enumeration methods in water and milk samples." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 58, no. 3 (June 2006): 432–39. http://dx.doi.org/10.1590/s0102-09352006000300023.
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Cryptosporidium spp. oocyst recovery in water and milk samples was evaluated. Samples were inoculated with a suspension of 1.2×10(7) Cryptosporidium spp. oocysts and submitted to centrifugal flotation, using different solutions (sucrose, NaCl, MgSO4, ZnSO4, AlSO4, NH4SO4 40% and NH4SO4 80%). Centrifugation of the samples was carried out in two stages for concentration using two methods that differed in the order in which the saturated solutions were used, namely only in the first stage of method I and only in the second stage of method II. Oocyst identification was performed using the Kinyoun and Koster histochemical staining techniques. Samples analyzed by method I showed different degree of oocyst recovery, namely 10.9% with NaCl and 42.5% with MgSO4 in water and milk samples, while those samples analyzed by method II showed 10.6% with NaCl and 5.3% with sucrose in water and milk, respectively. Histochemical staining methods have no influence on the degree of oocysts recovery. The efficiency of Cryptosporidium spp. oocysts recovery methods depends on the nature and composition of the sample and on the methodology used for oocyst concentration.
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Medema, G. J., M. Bahar, and F. M. Schets. "Survival of cryptosporidium parvum, escherichia coli, faecal enterococci and clostridium perfringens in river water: influence of temperature and autochthonous microorganisms." Water Science and Technology 35, no. 11-12 (June 1, 1997): 249–52. http://dx.doi.org/10.2166/wst.1997.0742.
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Oocysts of Cryptosporidium parvum can survive for several months in surface water, one of the main factors determining their success in environmental transmission and thus their health hazard via water. Several factors in the environment, e.g. temperature, presence of predators and exo-enzymes will probably influence oocyst survival. The high persistence of oocysts may also limit the value of traditional faecal indicator bacteria. The aim of this study was to determine the rate at which C parvum oocysts, E coli, faecal enterococci and C perfringens spores die in surface water and the influence of temperature and the presence of autochthonous (micro)organisms on the die-off rate. Microcosms with autoclaved river water were inoculated with the organisms. Microcosms with untreated river water were inoculated with concentrated primary effluent containing the bacteria and with C parvum oocysts. Microcosms were incubated at 5°C or 15°C at 100rpm. Viability of oocysts was monitored by in vitro excystation and dye-exclusion; viability of the bacteria was determined on appropriate selective media. When pseudo first-order die-off kinetics were assumed, the die-off rate of oocysts at 5°C was 0.010 log10/d and at 15°C, 0.006–0.024 log10/d. These rates underestimate die-off since oocyst disintegration was not accounted for. Incubation in autoclaved or untreated water did influence the die-off rate of oocysts at 15°C but not at 5°C. The die-off rate of E coli and enterococci was faster in the non-sterile river water than in autoclaved water at both temperatures. At 15°C, E coli (and possibly E faecium) even multiplied in autoclaved water. In untreated river water, the die-off of E coli and enterococci was approximately 10x faster than die-off of oocysts but die-off rates of C perfringens were lower than those of oocysts. As for oocysts, die-off of the bacteria and spores was faster at 15°C than at 5°C. Oocysts are very persistent in river water: the time required for a 10x reduction in viability being 40–160d at 15°C and 100d at 5°C. Biological/biochemical activity influenced oocyst survival at 15°C and survival of both vegetative bacteria at 5 and 15°C. The rapid die-off of E coli and enterococci makes them less suitable as indicators of oocyst presence in water. As C perfringens survived longer in untreated river water than oocysts, it may prove useful as an indicator of the presence of C parvum.
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Hirata, T., and A. Hashimoto. "Experimental assessment of the efficacy of microfiltration and ultrafiltration for Cryptosporidium removal." Water Science and Technology 38, no. 12 (December 1, 1998): 103–7. http://dx.doi.org/10.2166/wst.1998.0515.
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In order to evaluate the efficacy of microfiltration and ultrafiltration for Cryptosporidium oocyst removal, a bench-scale experiment was carried out using two 0.2m2 molecules, MF (nominal pore size 0.25μm) and UF (nominal cut-off MW 13,000 daltons) in cross-flow mode at an oocyst level of 106/L. Both of the membranes eliminated the oocysts from the influents with removal efficiency estimated to be >7 log10. As for the MF, an additional experiment was conducted at a much higher oocyst level up to 108 oocysts/L in both cross-flow and dead-end modes and which achieved >7 log10 removal, although some oocysts appeared in the filtrate in both modes. Based on these results, microfiltration and ultrafiltration are conclusively considered to be excellent processes for drinking water treatment as a single process that produces safe (an annual risk 10−4) water from highly polluted source waters.
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Graczyk, Thaddeus K., Ronald Fayer, Michael R. Cranfield, and David Bruce Conn. "Recovery of Waterborne Cryptosporidium parvum Oocysts by Freshwater Benthic Clams (Corbicula fluminea)." Applied and Environmental Microbiology 64, no. 2 (February 1, 1998): 427–30. http://dx.doi.org/10.1128/aem.64.2.427-430.1998.
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ABSTRACT Asian freshwater clams, Corbicula fluminea, exposed for 24 h to 38 liters of water contaminated with infectiousCryptosporidium parvum oocysts (1.00 × 106 oocysts/liter; approximately 1.9 × 105 oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used—acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MERIFLUOR test kit).