A series of pilot-scale granular media filtration experiments was conducted to examine the effect of media roughness on filter performance and to evaluate the applicability of spherical, rough engineered ceramic filter media for use in granular media filters used for drinking water treatment. Filter media performance was assessed using turbidity and particle count reductions, Cryptosporidium oocyst and oocyst-sized microsphere removal, head loss and stability of operation. Experiments were designed to allow related facets of current filtration research to be examined. These included: effect of loading rate, coagulant type and dosage, and suitability of latex microspheres as surrogates for Cryptosporidium oocyst removal by granular media filtration. This study indicated that increased filter media roughness consistently improved turbidity and particle count reduction under the conditions investigated. As well, the engineered media also consistently achieved greater stability of operation during non-ideal operational periods (e.g. sudden change in filter influent turbidity).Oocyst removals were generally improved by media roughness, though this improvement was reliant on operating conditions, such as coagulant dose and type of coagulant used. The surrogate relationship between oocyst-sized latex microspheres and oocyst removal by filtration was also dependent on coagulant dose and type of coagulant. During trials with no coagulant addition, contrasts in oocyst removal were not significant, suggesting that neither surface roughness nor the size of media used were significant factors impacting oocyst removal by filtration during those periods of impaired operation. When pre-treating raw water with PACl, the engineered ceramic media achieved up to 1.25 log10 higher oocyst removals than conventional media. This improvement in oocyst removal relative to conventional media was not observed when alum was used as the primary coagulant, however. Future studies should directly compare engineered and conventional media filtration performance, using other raw water sources and different operating conditions. Biologically active filtration should also be included in future performance studies because the rough, highly porous surface of the engineered ceramic media is likely to provide excellent biofilm support.

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

Protozoa and bacteria can easily cause disease in humans, specifically E. coli, Plasmodium falciparum, and Cryptosporidium parvum. These three pathogens are associated with large public health concerns that span the globe. The variety of locations in which these can be found is extremely high. Cryptosporidium spp. are extremely resilient when in oocyst form, P. falciparum is in the Anopheles spp. mosquito, while E. coli can be found on anything from food and water, to the skin and gut. The diverse range of locations these can be found in means that a portable sensor for their detection is necessary. In detecting Cryptosporidium, microscopy is the preferred method of identification currently. This requires a trained lab technician as well as calibrated and expensive optical equipment. Technician error can lead to false negative or positive diagnoses as well as sample destruction. A method to remove this technician interaction is thus necessary. This method must allow for objective results that are not open to interpretation. Particle immunoagglutination assays with Mie scatter allow for such an approach using inexpensive components. Particle immunoagglutination relies on the principles of antibody-antigen interaction and antibody conjugated latex particles. Using highly carboxylated latex particles, it is possible to attach IgG antibodies that are specific to a target antigen. Mie scatter is governed by particle size rather than wavelength as other forms of scatter. These two combined allow for an increase in light scatter based on particle size. This is correlated in a linear manner as long as the number of antibody-conjugated particles is higher than the number of antigens. Microfluidics is an ever growing field in the field of lab-on-a-chip that works very well with particle immunoagglutination. In this paper, a method to rapidly identify the presence of Cryptosporidium using microfluidics and particle immunoagglutination is discussed and analyzed. This method allows for a low detection limit of 1-10 oocysts/sample and an assay time of approximately 10 minutes. Results are displayed on a computer screen as the value of light scatter intensity and, when compared to a standard curve, is an objective way to identify the concentration and presence of oocysts in a diverse range of samples. These samples include PBS, pool water, and sump water. This system also works with P. falciparum, which causes malaria in rural and urban poor regions of the world. With the low income and remote nature of these locations, a portable microfluidic device is necessary. Smartphones allow for a portable microfluidic device that can detect P. falciparum antigens in 10% whole blood. This system is capable of detecting as little as 1 pg/mL antigen. The microfluidic chip is inexpensive and disposable, allowing for a portable and inexpensive system. Using a single smartphone, a lab technique requiring a spectrometer, light source, and laptop can be made portable and less expensive, while maintaining sensitivity and specificity. In order to identify biological agents, there are commonly 3 methods for doing so: PCR, culturing, and ELISA. Culturing can take more than 24 hours, but results in a high signal to initial target ratio, while ELISA has poor sensitivity due to a 1:1 signal to target ratio, though is much quicker than culturing at usually 3 hours or less. PCR manages to solve both these problems by exponentially increasing the number of copies of target genetic material in a relatively short time frame of 1-3 hours. PCR relies on 4 basic components: target genetic material, primers to set a start and end location for duplication, polymerase to add base pairs to the strand beginning at the primers, and heat. PCR has worked very well during the past 31 years. It has worked so well that it is often the gold standard. However, there are flaws built into today's systems. These largely come in the form of inefficient heat transfer via conduction and large sample volumes due to unnecessary additions of nuclease free water (NFW). Both of these can be easily overcome by droplet PCR. Droplet PCR relies on small sample volumes of between 8 and 12 μL and convection in oil rather than conduction through plastic. In this study, it was found that droplet PCR could be performed on genomic E. coli DNA in as little as 15 minutes for 30 cycles. Sensitivity was also analyzed and found to be 2.62 pg DNA/μL or about 5 x 10² cfu/sample. PCR has a theoretical lower limit of 1 copy of genetic material and this is only 2 orders of magnitude above that. The system was also tested for portability and resistance to shock and vibration. It was found that the surface heated, thermocouple guided system is more shock and vibration resistant than standard wire guided, hanging droplet PCR systems. It was also found that the use of coconut oil allows for the system to be transported without fear of the contents spilling out and contaminating other samples. This is because of coconut oil's high melting temperature.

No description available.

Water supply systems aim, among other objectives, to protect public health by reducing the concentration of, and potentially eliminating, microorganisms pathogenic to human beings. Yet, because water supply systems are engineered systems facing variable conditions, such as raw water quality or treatment process performance, the quality of the drinking water produced also exhibits variability. The reliability of a treatment system is defined in this context as the probability of producing drinking water that complies with existing microbial quality standards. This thesis examines the concept of reliability for two physicochemical treatment technologies, conventional rapid granular filtration and ultrafiltration, used to remove the protozoan pathogen Cryptosporidium parvum from drinking water. First, fault tree analysis is used as a method of identifying technical hazards related to the operation of these two technologies and to propose ways of minimizing the probability of failure of the systems. This method is used to compile operators’ knowledge into a single logical diagram and allows the identification of important processes which require efficient monitoring and maintenance practices. Second, an existing quantitative microbial risk assessment model is extended to be used in a reliability analysis. The extended model is used to quantify the reliability of the ultrafiltration system, for which performance is based on full-scale operational data, and to compare it with the reliability of rapid granular filtration systems, for which performance is based on previously published data. This method allows for a sound comparison of the reliability of the two technologies. Several issues remain to be addressed regarding the approaches used to quantify the different input variables of the model. The approaches proposed herein can be applied to other water treatment technologies, to aid in prioritizing interventions to improve system reliability at the operational level, and to determine the data needs for further refinements of the estimates of important variables.

Das Ziel der Arbeit war, ein In-vitro-Infektionsmodell für den protozoären Parasiten C. parvum zu etablieren, um eine Aussage über die Infektiosität der Oozysten treffen zu können. Es wurde ein robustes Zellkultursystem zur In-vitro-Kultivierung dieses Einzellers erarbeitet. Die Oozysten von C. parvum wurden im neonatalen Kälbern passagiert und aus dem Kot isoliert und aufgereinigt, so dass ein direkt in die Zellkultur einsetzbares Inokulum gewonnen werden konnte. Als Zelllinie wurden adhärent wachsende HCT-8-Zellen verwendet. Die konfluenten Zellmonolayer wurden in Mikrotiterplatten direkt mit der Oozystensuspension inokuliert. Dabei wurde dem Medium zur Erleichterung der Exzystierung der Sporozoiten 0,4 % Natriumtaurocholat beigemischt, ohne dass dabei ein zytotoxischer Effekt festzustellen war. Die erfolgreiche Exzystierung und Invasion der Zellen durch die Parasiten mit anschließender Vermehrung konnte bei hohen Anzahlen inokulierter Oozysten mit einer geeigneten Kontrasttechnik mikroskopisch beobachtet werden. Zum sicheren semiquantitativen Nachweis der Infektion wurde ein PCR-basierter Assay verwendet. Die Robustheit und Sensitivität des Zellkultur-PCR-Systems wurde mit vitalen Oozysten getestet und anschließend auf die Testung des Einflusses einer physikalischen (hohe Temperatur) und chemischen (Neopredisan®) Inaktivierungsmethode auf die Vitalität der Oozysten angewandt. Es wurden insgesamt 271 Zellkulturen mit unterschiedlichen Mengen vitaler Oozysten inokuliert. Mindestens 1000 Oozysten waren notwendig, um eine sicher nachweisbare Infektion zu erzeugen. Bei Einsatz von 100 Oozysten konnten etwa drei von vier Kulturen als infiziert diagnostiziert werden, bei Verwendung von 10 Oozysten betrug dieser Anteil etwas unter einem Drittel. Bei der thermischen Inaktivierung wurden zwei Temperaturen benutzt: 38 °C und 55 °C, die für unterschiedlich lange Zeitspannen (zwischen 1 und 24 h) auf die Oozysten eingewirkt hatten. In den 68 so untersuchten Kulturen zeigte sich, dass 55 °C unabhängig von der Einwirkdauer ausreichend waren, um die Oozysten soweit zu inaktivieren, dass kein PCR-Nachweis in der Kultur mehr möglich war. Eine Erwärmung auf lediglich 38 °C hatte dagegen keinen nennenswerten Einfluss auf die Infektiosität der Oozysten in der Zellkultur. Diese Oozysten verhielten sich ähnlich wie die unbehandelten Chargen. Es wurde ferner der Einfluss einer einstündigen Exposition mit Neopredisan® in den Konzentrationen von 0,25, 1 und 4 % auf die Infektiosität getestet (80 untersuchte Kulturen), sowie einer zweistündigen Inkubation mit 4 % (60 PCR-Ansätze). Die beiden niedrigen Konzentrationen übten keinen hemmenden Effekt auf die Vermehrungspotenz der so behandelten Oozysten aus, teilweise wurde hier sogar eine erhöhte Nachweisbarkeit vor allem in den niedrigen Inokula festgestellt. Dagegen konnte die höchste Konzentration die Oozysten in ihrer Vermehrung signifikant hemmen. Die längere Desinfektion erwies sich hierbei als wirksamer. Dieser Effekt war jedoch deutlich geringer als der einer Erwärmung auf 55 °C, und es konnten nicht alle Oozysten inaktiviert werden. Die Genotypisierung (Sequenzierung von zwei Genloci) der verwendeten Isolate ergab, dass es sich bei den verwendeten Parasiten um den bovinen Genotyp von C. parvum handelte und dass durch die verwendete PCR der richtige DNA-Abschnitt amplifiziert wurde. Insgesamt gesehen ist die Methode geeignet, reproduzierbare intrazelluläre Infektionen einer permanenten Zelllinie in vitro zu erzeugen, die sich auch gut mit der vorgestellten PCR nachweisen lassen. Allerdings ist auf diese Weise nur eine semiquantitative Abschätzung der Intensität der Entwicklung (über eine Titrationsreihe) möglich. Es wäre anstrebenswert, hier eine quantitative Methode (z.B. quantitative PCR) einsetzen zu können.

In dieser Arbeit wird eine neue Methode der Vitalitätsbestimmung von Cryptosporidium parvum-Oozysten beschrieben. Die gereinigten Oozysten wurden in einer HCT-8-zelllinie kultiviert und mittels IFAT ausgewertet. Um eine genaue Quantifizierung der fluoreszierenden Flächen vornehmen zu können, wurden die Bilder einer Bildanalysesoftware zugeführt und analysiert. Die Menge eingesäter Oozysten korrelierte signifikant mit den gemessenen Flächen intrazellulärer Entwicklungsstadien. In diesem System wurden verschiedene Feldisolate vergleichend getestet sowie die Vitalität thermisch inaktivierter Oozysten bestimmt.

Die Arbeit hatte zum Ziel, ein In-vitro-Infektionsmodell für den protozoären Durchfallerreger Cryptosporidium parvum zu optimieren und dahingehend zu testen, ob es für eine quantitative Beurteilung der Infektiosität von Kryptosporidienoozysten eingesetzt werden kann. Die verwendeten Oozysten wurden zuvor im Zuge einer Passagierung im Kalb vermehrt, aus dem Kot isoliert, aufgereinigt und zur Infektion einer humanen ileocaecalen Adenokarzinomzelllinie (HCT-8) verwendet Die Kultivierung erfolgte über 48 Stunden in Mikrotiterplatten mit jeweils 24 Kavitäten. Die DNA infizierter Zellen und nichtinfizierter Kontrollen wurde anschließend isoliert und die parasitenspezifische DNA in der real-time PCR quantifiziert. Die gewählten Primer-Sonden-Kombinationen erlaubten eine spezifische Amplifikation der Erreger-DNA. In der Optimierung wurden das Brilliant®QPCR Core Reagent Kit, der ABsoluteTMQPCR sowie zwei verschiedene Oligonukleotidkombinationen untersucht. Durch die Klonierung einer Sequenz im Target-Gen und die Herstellung einer Titrationsreihe aus dieser klonierten DNA gelang es, den für die Vergleichbarkeit unerlässlichen homogenen Standard zu gewinnen. Der In-vitro-Vitalitätsassay wurde außerdem auf seine praktische Anwendbarkeit hin geprüft. Es wurde einerseits eine Desinfektionsmittelprüfung mit Chlorokresol (Neopredisan®135-E), andererseits ein Versuch zur thermischen Inaktivierung, beide unter Nutzung dreier verschiedener C. parvum-Chargen (LE-06-Cp-05/0, LE-07-Cp-05/2 vom Isolat A, LE-06-Cp-05/2 vom Isolat B), vollzogen. Die Überbewertung der Infektiosität der Oozysten durch die Betrachtung der Exzystierung konnte anhand der parallel zur DNA-Quantifizierung ermittelten Exzystierungsraten gezeigt werden. Die Exzystierungshemmung lag in jedem Versuch deutlich unter den in der real-time PCR berechneten Inaktivierungsraten. Je nach verwendeter Oozystencharge lieferte die Desinfektion mit 4 % Neopredisan®135-E Inaktivierungsraten, die zwischen 90 und 100 % bei einstündiger Einwirkzeit lagen. Mit steigender Dauer der Inkubation stieg erwartungsgemäß auch der Grad der Inaktivierung. Die Anwendung der 1 %igen Verdünnung resultierte in einer deutlich gesteigerten Exzystierungsrate gegenüber der unbehandelten Kontrolle sowie in stark variierenden Inaktivierungsraten (24 - 91,5 %). Es konnte gezeigt werden, dass mit Neopredisan®135-E unter den gewählten Inkubationsbedingungen zwar eine gute, aber keine vollständige Inaktivierung der C. parvum-Oozysten erfolgt. Eine suboptimale Wirkung zeigte sich in einer hohen Varianz der Einzelmesswerte. Die Vitalitätsraten betrugen nach einstündiger Inkubation der Oozysten bei 38°C noch 100 %, nach 24 Stunden waren diese bereits auf 5 - 23 % abgesunken. Es scheint, als würden mesophile Verhältnisse die Exzystierung der Sporozoiten anregen und bei längerer Konditionierung eine Erschöpfung des Stoffwechsels der Entwicklungsstadien herbeiführen. Die Inaktivierungsrate bei 55°C lag zwischen 96 und 100 %. Bei thermophiler Konditionierung wurde in drei von sieben Fällen, nach der Inkubation in Neopredisan®135-E nur in einer der sieben Untersuchungen ein vollständiger Vitalitätsverlust beobachtet. Die vorgestellte Methode erwies sich als gut reproduzierbar, sensitiv und schnell. Die In-vitro-Kultivierung des Erregers C. parvum ließ sich mit der real-time PCR, welche eine absolute Quantifizierung erlaubte, gut in Einklang bringen. Die Verwendung der In-vitro-Kultur als lebendes System ließ eine gewisse Variabilität der Ergebnisse zwischen einzelnen Untersuchungen erwarten, die sich aber in einem akzeptablen Bereich bewegten. Eine weitere Optimierung im Sinne einer Sensitivitätssteigerung bei akzeptabler Störanfälligkeit und Variabilität ist anzustreben.

Cryptosporidium parvum is an obligatory intracellular parasitic protist that belongs to the phylum Apicomplexa. Cryptosporidiosis is an infection for which no satisfactory efficient curative treatment is known, especially in immunocompromised individuals. Furthermore, the parasite oocysts show considerable tenacity in the environment. Therefore, new potent drugs along with a simple and reliable experimental model for evaluation of anticryptosporidial measures are urgently needed. The present studies were undertaken to establish a combined cell culture and quantitative PCR assay (cc-qPCR) to assess efficacy of pharmacological compounds against C. parvum. Human ileocecal adenocarcinoma cells (HCT-8) were selected for culture of C. parvum. Oocysts were excysted directly on confluent monolayers for infection. After 3 h of incubation the non invasive parasite remains were removed by washing. At the end of the incubation period the cells were harvested and subjected to DNA extraction. Real time PCR was performed to quantify the target parasite DNA (fragments of 70 kDa heat shock protein gene) copy numbers. Each reaction was run in triplicate. A standard curve calculated on the basis of serial dilutions of plasmid DNA or infected control culture DNA was run in each experiment. A series of oocyst suspensions were applied to cell cultures to determine the sensitivity of the cc-qPCR assay and also to generate a calibration curve to calculate the infectivity of oocysts. A dilution series of heat inactivated oocysts (70°C for 1 h) were used to determine the size of the oocyst inoculum at which complete elimination of extracellular parasite material by washing is reliably achieved. The results obtained by the assays were reproducible and the method sensitive with a detection limit of infection with 10 oocysts 48 h post infection (p.i.) and with 100 oocysts 24 h p.i. Percent effects of drugs and disinfectants were enumerated by comparing DNA copies between treated and non treated samples. The suitability of cc-qPCR for screening of pharmacological compounds was validated by confirming the in vitro efficacy of monensin (98.15% ± 1.09 at 0.144 µM) and halofuginone (98.05% ± 0.59 at 25 µM) over the entire incubation period with a dose dependent reduction of parasite multiplication demonstrated 27 h p.i. The inhibition of parasite proliferation by 0.144 µM monensin in the period from 3 h p.i (time defined to represent the initial level of parasite development before drug application) to 27 h p.i. or 45 h p.i. was 97 and 99% respectively, and by 25 µM halofuginone 99% (27 h p.i.). Hexadecylphosphocholine (miltefosine), a new anti-leishmanial compound, was tested against cryptosporidia and provided a maximum of 98% reduction of parasite multiplication at 45 h p.i. The potential activity of curcumin (extract from the herb Curcuma longa) against C. parvum was also evaluated by cc-qPCR. Curcumin appeared to be sensitive to degradation after prolonged incubation and the observed inhibition of multiplication of C. parvum was significantly increased when medium was replaced by fresh medicated medium after 12 h of exposure. The effects on parasite multiplication (>95% inhibition with IC50 value of 13 µM) and on sporozoite invasion (assessed 3 h p.i.; 65% inhibition at 200 µM) suggest that further exploration of anticryptosporidial efficacy of curcumin may be rewarding. The cc-qPCR was further optimized to analyse inactivation measures directed against oocysts of C. parvum. The suitability of the assay for assessment of inactivation measures was confirmed by the reproducible demonstration of effectiveness of cresolic disinfectants at the recommended concentration of 4% and incubation period of 2 h (Neopredisan® 135-1, Menno Chemie, Norderstedt, Germany: 99.91% ± 0.08; Aldecoc® TGE, EWABO Chemikalien GmbH & Co. KG, Wietmarschen, Germany: 99.91± 0.05) and by using thermally inactivated oocysts (complete inactivation by 56°C and 70°C for 20 min). Based on the in vitro results and previously obtained data from the chicken infection model 99.5% inactivation is proposed as a suitable threshold value that needs to be consistently exceeded by a product to be considered efficient. Application of Neopredisan® 135- 1 and Aldecoc® TGE (4% for 2h) consistently inactivated more than 99.5% of oocysts while other disinfectants that are not certified as anticoccidial products like Aldecoc® XD (EWABO Chemikalien GmbH & Co. KG, Wietmarschen, Germany) and IGAVET® FF spezial (COS OHLSEN Chemie & Gerätevertrieb GmbH, Geltorf-Esprehm, Germany) and bleach (sodium hypochlorite) did not. It can be concluded that the cc-qPCR method is suited to easily and reliably assess anticryptosporidials in vitro. The method demonstrated that miltefosine and curcumin display anticryptosporidial efficacy under the applied conditions. The cc-qPCR is a highly standardized method supposedly appropriate to replace the chicken infection model for Eimeria tenella as currently practised for certification of anticoccidial disinfectants according to the guidelines of DVG (German Veterinary Society).

Water supply systems aim, among other objectives, to protect public health by reducing the concentration of, and potentially eliminating, microorganisms pathogenic to human beings. Yet, because water supply systems are engineered systems facing variable conditions, such as raw water quality or treatment process performance, the quality of the drinking water produced also exhibits variability. The reliability of a treatment system is defined in this context as the probability of producing drinking water that complies with existing microbial quality standards. This thesis examines the concept of reliability for two physicochemical treatment technologies, conventional rapid granular filtration and ultrafiltration, used to remove the protozoan pathogen Cryptosporidium parvum from drinking water. First, fault tree analysis is used as a method of identifying technical hazards related to the operation of these two technologies and to propose ways of minimizing the probability of failure of the systems. This method is used to compile operators’ knowledge into a single logical diagram and allows the identification of important processes which require efficient monitoring and maintenance practices. Second, an existing quantitative microbial risk assessment model is extended to be used in a reliability analysis. The extended model is used to quantify the reliability of the ultrafiltration system, for which performance is based on full-scale operational data, and to compare it with the reliability of rapid granular filtration systems, for which performance is based on previously published data. This method allows for a sound comparison of the reliability of the two technologies. Several issues remain to be addressed regarding the approaches used to quantify the different input variables of the model. The approaches proposed herein can be applied to other water treatment technologies, to aid in prioritizing interventions to improve system reliability at the operational level, and to determine the data needs for further refinements of the estimates of important variables.